bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Structure and chromosomal assignment of a gene encoding the major protein of rat sperm outer dense fibres
Authors: BURFEIND Peter , BELGARDT Bettina , SZPIRER Claude , HOYER‐FENDER Sigrid
Published: March 2005

Outer dense fibres are located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail. Here we describe the isolation and genomic organization of a rat gene encoding a cysteine‐proline‐rich outer dense fibre protein. The cDNA sequence of rts 5/1 and its expression pattern have already been published [Burfeind, P. & Hoyer‐Fender, S. (1991) Dev. Biol. 148, 195–204]. There exist two different genes in the rat genome, rts 5/1 major and rts 5/1 minor. Rts 5/1 major consists of two exons interrupted by one intron of about 3.8 kb. Exon 1 of rts 5/1 minor contains a deletion of 120 bp, without destroying the open reading frame, which is flanked by short direct repeats, 15 bp in lenght. The first two nucleotides of the intronic sequence were identified as GA and, therefore, do not agree with the donor consensus sequence. From the analysis of mouse × rat cell hybrids, the rts 5/1 major gene has been assigned to chromosome 7. By immunoblotting and immunocytochemistry, it was demonstrated that the isolated gene encodes a major protein of rat sperm outer dense fibres.
Exploration of the single‐stranded DNA‐binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin‐labeled oligonucleotide and lanthanide‐chelate complexes
Authors: VAN DUYNHOVEN John P. M. , NOOREN Irene M. A. , SWINKELS Dorine W. , FOLKERS Paul J. M. , HARMSEN Ben J. M. , KONINGS Ruud N. H. , TESSER Godefridus I. , HILBERS Cornelis W.
Published: March 2005

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a β‐sheet structure which is partly five stranded. The DNA‐binding domain of IKe GVP was investigated using a spin‐labeled deoxytrinucleotide. The paramagnetic‐relaxation effects observed in the 1H‐NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two‐dimensional differnce spectroscopy. In this way, the residues present in the DNA‐binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin‐labeled oligonucleotide. Binding studies with negatively charged lanthanide‐1,4,7,10‐tetraazacyclodecanetrayl‐1,4,7,10‐tetrakis(methylene) tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high‐affinity binding site for Gd(DOTP)5−/M13 GVP monomer, which coincides with the single‐stranded DNA‐binding domain as mapped with the aid of spin‐labeled oligonucleotide fragments. The results show that single‐stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)5−‐binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrphobic residues in the cores of the M13 and IKe dimers.
Acylation of porcine pancreatic phospholipase A2 influences penetration and substrate head‐group binding, depending on the position of the acylated lysine in the enzyme molecule
Authors: LUGTIGHEID Richard B. , NICOLAES Gerrie A. F. , VELDHUIZEN Edwin J. A. , SLOTBOOM Arend J. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

A porcine pancreatic phospholipase A2 mutant was constructed in which all nine lysines were replaced by arginines. The mutant displayed 68% residual activity on micellar zwitterionic substrates, indicating that lysines are not absolutely required for the catalytic action of the enzyme. Likewise, mutants with one single lysine present either at position 56, located close to the entrance of the active site, or at position 108, remote from the active site, were constructed. Selective acylation of Lys56 with acyl chains of two, eight or fourteen carbon atoms resulted in increased activities on 1,2‐dioctanoylglycero‐3‐phosphocholine micelles. Moreover, acylation strongly influenced the affinity for these micelles, as was evidenced by an up to 60‐fold increase in apparent Km. The kinetic properties of the (acylated) mutants were studied with the monolayer technique. Pre‐steady‐state kinetics showed that penetration into monomolecular layers composed of 1,2‐didodecanoylglycero‐3‐phosphocholine was faster for acylated Lys56 derivatives than for non‐acylated enzyme. The acylated enzymes were also capable of penetrating densely packed lipid films. This effect increased with increasing acyl chain length. The observed velocities in the steady state were similar for acylated and non‐acylated Lys56 mutants. In contrast, no changes in the kinetic properties were observed after acylation of Lys108, located on the posterior part of the protein. Therefore, the effects observed upon acylation of Lys56 are probably specific. Apart from an increase in hydrophobicity, acylation of Lys results in charge neutralization. The latter effect was studied with a mutant in which Gln instead of Lys was present at position 56. The activity of this mutant on micellar substrates is higher than that of the parent Lys56, whereas its affinity for micelles is slightly improved. Therefore, whereas the charge at position 56 mainly influences the activity, the hydrophobicity of the introduced acyl chain mainly determines the affinity for aggregated lipids.
Molecular assembly of plasminogen and tissue‐type plasminogen activator on an evolving fibrin surface
Authors: FLEURY Valérie , LOYAU Stéphane , LIJNEN H. Roger , NIEUWENHUIZEN Willem , ANGLÉS‐CANO Eduardo
Published: March 2005

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue‐type plasminogen activator (t‐PA), plasminogen and fibrin. The molecular assembly of t‐PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di‐isopropylphosphofluoridate‐inactivated t‐PA, and a recombinant human plasminogen with the active‐site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t‐PA, indicating that binding sites for plasminogen pre‐exist in intact fibrin and are not dependent on the presence of t‐PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r= 0.98) to the appearance of the fibrin E‐fragment as shown using a monoclonal antibody (FDP‐14) that has its epitope in the E domain of fibrin. t‐PA was shown to bind with a high affinity to both the intact (Kd= 3.3 ± 0.6 nM) and the degraded surface of fibrin (Kd= 1.2 ± 0.4 nM). Binding of t‐PA to carboxy‐terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 μM), indicating that the affinity of t‐PA for these residues was lower than that of plasminogen (Kd= 0.66 ± 0.22 μM) and unrelated to the high affinity of t‐PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy‐terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.
Cholesterol trafficking in steroidogenic cells
Authors: STEVENS Victoria L. , XU Tingsen , LAMBETH J. David
Published: March 2005

Peptide hormones activate steroid hormone biosynthesis in responsive tissues by stimulating the delivery of cholesterol to a steroidogenic pool, thought to be located in the inner mitochondrial membrane. At this site, it is metabolized to pregnenolone, the precursor of the steroid hormones, by side‐chain‐cleaving cytochrome P‐450 (cytochrome P‐450scc). In the presence of aminoglutethimide (an inhibitor of cytochrome P‐450scc) and an activating stimulus, cholesterol accumulates in the steroidogenic pool, and increased pregnenolone generation is observed upon removal of the inhibitor. Using Y‐1 adrenocortical cells and MA‐10 Leydig tumor cells, we now provide evidence for a distinct, functionally relevant cholesterol pool which precedes the steroidogenic pool, which we designate the pre‐steroidogenic pool. This pool was defined by activating the cells with 8‐bromo‐adenosine 3′,5′‐cyclic monophosphoric acid in the presence of cycloheximide, an inhibitor of steriodogenesis. Following a wash procedure, which removed 8‐bromo‐adenosine 3′,5′‐cyclic monophosphoric acid and cyclohcximide, augmented pregnenolone synthesis was observed. Unlike synthesis from the steroidogenic pool, pregnenolone formation from pre‐steroidogenic pool in Y‐1 cells indicates that this pool is somewhat smaller than the steroidogenic pool. The results support a cholesterol‐trafficking model in which cycloheximide‐sensitive transport from the pre‐steroidogenic pool to the steroidogenic pool precedes metabolism, and is regulated by cAMP.
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.
Activation and substrate specificity of the human protein kinase C α and ζ isoenzymes
Authors: KOCHS Georg , HUMMEL Richard , MEYER Diana , HUG Hubert , MARMÉ Dieter , SARRE Thomas F.
Published: March 2005

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub‐groups: the Ca2+ ‐dependent (classical) PKC isoenzymes α, βI, βII and γ, and the Ca2+ ‐independent neoPKC isoenzymes δ, ε, ζ, ν and θ. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction.

In this study, we report the cloning of a human PKC‐ζ cDNA, its expression in recombinant baculovirus‐infected insect cells and the partial purification of the PKC‐ζ isoenzyme. In comparison to higly purified human PKC α, a representative of the classical PKC subgroup, purified PKC ζ was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC α, PKC ζ exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of γ‐linolenic acid and phosphatidylserine slightly enhance PKC ζ activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC α phosphorylates a PKC‐α pseudosubstrate‐derived peptide, an epidermal‐growth‐factor‐receptor‐derived peptide, histone III‐S and myelin basic protein to an equal extent, whilst PKC ζ phosphorylates only the PKC‐α‐derived peptide. However, arachidonic acid greatly diminishes PKC‐α activity towards the epidermal‐growth‐factor‐receptor‐derived peptide, histone III‐S and myelin basic protein, but enhances PKC‐ζ activity towards the PKC‐α‐derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC ζ, this finding is strengthened by the fact that the epidermal growth factor receptor‐derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC ζ, in contrast to PKC α, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) activation and the substrate specificity of PKC ζ are discussed.
Acyl‐CoA synthetase mRNA expression is controlled by fibric‐acid derivatives, feeding and liver proliferation
Authors: SCHOONJANS Kristina , STAELS Bart , GRIMALDI Paul , AUWERX Johan
Published: March 2005

Several enzymes of the ß‐oxidation pathway have been shown to be induced after stimulation with peroxisomal proliferators, including several hypolipidemic drugs. We investigated the regulation of the long‐chain‐acyl‐CoA synthetase (ACS) gene in the liver. Fenofibrate, a hypolipidemic drug and potent peroxisomal proliferator, induced ACS gene expression in several tissues. In liver, large increases in ACS mRNA levels and ACS activity were observed after fenofibrate administration. Adipose tissue ACS mRNA levels and ACS activity were also stimulated upon fibrate treatment but to a lesser extent in comparison with liver ACS mRNA. Kidney ACS mRNA was only weakly induced, except for the highest dose and the longest treatment period, where a strong induction was observed. In contrast to these tissues, heart ACS mRNA and ACS activity remained almost unchanged after fenofibrate treatment. These effects of fenofibrate could be reproduced by other fibrates such as clofibrate. In addition, it is demonstrated that both nutritional composition and liver proliferation trigger ACS gene expression in liver. Consequently, these data suggest that ACS is a highly regulated enzyme with a potentially important control function in lipid metabolism.
Purification and characterization of new anti‐adrenocorticotropin rabbit neutrophil peptides (defensins)
Authors: FUSE Naoyuki , HAYASHI Yoshio , FUKATA Junichi , TOMINAGA Tomoko , EBISUI Osamu , SATOH Yoshimi , ISOHARA Toshio , UNO Isao , IMURA Hiroo
Published: March 2005

Neutrophil peptides (NPs, defensins), which consist of approximately 30 amino acids with a highly conserved backbone of six Cys residues, possess several biological activities, such as antimicrobial, antiviral, cytotoxic, and anti‐adrenocorticotropin (corticostatic) activity in vitro. In the rabbit, six NPs, i.e., NP‐1, ‐2, ‐3A, ‐3B, ‐4, and ‐5, have been isolated, among them, NP‐3A has the most potent corticostatic activity, which involves the inhibition of adrenocorticotropin‐stimulated corticosterone synthesis in adrenal cells. Using a sensitive radioimmunoassay for NP‐3A and reverse‐phase HPLC, we found a novel component in rabbit tissue extracts with NP‐3 A‐like immuno‐reactivity. We further purified and characterized the component and found two novel peptides. One of these peptides, designated NP‐6, has an amino acid sequence identical to that of NP‐3A except for three amino acids, i.e. Leu11, Phe13, and Gln15, in the central portion of the sequence. The other has a sequence corresponding to that of NP‐6 except that the N‐terminal Gly was deleted and is thus named des‐G1‐NP‐6. Using chemically synthesized NP‐6 and des‐G1‐NP‐6, we measured their putative corticostatic activities by a dispersed rat adrenal cell bioassay. NP‐6 showed corticostatic activity comparable to that of NP‐3 A while des‐G1‐NP‐6 showed weak activity. These findings are also compatible with the notion that the N‐terminal Gly, but not the central portion, is important for the anti‐adrenocorticotropin activity of the NPs.
The l‐lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli
Authors: OSTENDORP Ralf , LIEBL Wolfgang , SCHURIG Hartmut , JAENICKE Rainer
Published: March 2005

The gene for a l(+)‐lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. ldh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine‐Dalgarno sequence within the non‐coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme.

Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequenc̀e with those of known prokaryotic l‐lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found.

The gene was expressed as an active enzyme in a heterologous host.
Purification and primary structure of snail metallothionein
Authors: DALLINGER Reinhard , BERGER Burkhard , HUNZIKER Peter E. , BIRCHLER Neil , HAUER Charles R. , KÄAGI Jeremias H. R.
Published: March 2005

A cadmium‐binding metallothionein has been purified from metal‐exposed Roman snails (Helix pomatia) using gel‐permeation, ion‐exchange and reverse‐phase high‐performance liquid chromatorgraphy. The S‐methylated protein was digested with trypsin and the endoproteinases Asp‐N, Glu‐C and Arg‐C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N‐terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N‐terminal amino acid was an acetylated serine residue.

Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys‐Xaa‐Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N‐terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N‐terminal sequences of histones H2A and H4 which may be important for regulatory functions.
Probes of ligand‐induced conformational change in aspartate aminotransferase
Authors: GARNIER Annelise , JOHN Robert A.
Published: March 2005

Sodium borohydride and sodium cyanoborohydride were assessed as potential reagents for determining ligand‐induced changes in accessibility to the active‐site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determined in the presence of increasing concentrations of the dicarboxylate substrate analogues glutarate and maleate. The rate of reduction decreased to a limiting value which was about 40‐fold lower than the equivalent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompanied binding of dicarboxylates. Allowing for this increase, the true decrease in accessibility to NaBH3CN was estimated to be approximately 400‐fold. Arguments are presented in support of a proposal that the ratio of closed to open conformer of the dicarboxylate‐liganded enzyme is approximately 150. The effects of increasing ligand concentration on the reactivity of Cys390 were found to take place in the same range as was observed for NaBH3CN reduction. Conversely, very much higher concentrations of the dicarboxylates were required to protect against proteolysis by trypsin. It is concluded that NaBH3CN reduction and reactivity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an additional binding mode for the dicarboxylates.
The use of human milk fucosyltransferase in the synthesis of tumor‐associated trimeric X determinants
Authors: De VRIES Theodora , NORBERG Thomas , LÖNN Hans , VAN DEN EIJNDEN Dirk H.
Published: March 2005

We have studied the fucosylaticn of a chemically synthesized trimer of N‐acetyllactosamine [(LacNAc)3‐EtPhNHCOCF3] with a fucosyltransferase preparation from normal human milk, which utilizes both type‐1 and type‐2 structures, whether sialylated or not. When fucose residues were added enzymically to the (LacNAc)3‐EtPhNHCOCF3 hexasaccharide, mono‐, di‐, or trifucosylated oligosaccharide species were formed, containing the Lewisx determinant (Galβ1→4[Fucα1→3]GlcNAcβ1→3). With excess GDP‐fucose and prolonged reaction times, the trifucosylated product was formed in almost quantitative yield. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second and third fucose residues onto the acceptor molecule. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was assessed by analyzing the digests obtained after endo‐β‐galactosidase treatment by HPLC and reverse‐phase chromatography. In addition, the fucosylated (LacNAc)3‐EtPhNHCOCF3, structures were characterized by HPLC and were identified by 400‐MHz 1H‐NMR spectroscopy. There is a highly preferred order in which the fucosyl residues are attached to (LacNAc)3‐EtPhNHCOCF3. In the major pathway, the first two fucose residues are transferred with equal preference to the medial (GN3) and proximal (GN1) GlcNAc residues, whereas the third fucose is attached to the distal (GN5) GlcNAc residue. These results are of relevance in understanding the role of α‐3‐fucosyltransferase in the biosynthesis of Lewisx‐related cell‐surface carbohydrate structures, that function as ligands for selectin‐type cell‐adhesion molecules and may play a role in the invasion and metastasis of several carcinoma.
Studies on O‐glycans of Plasmodium‐falciparum‐infected human erythrocytes Evidence for O‐GlcNAc and O‐GlcNAc‐transferase in malaria parasites
Authors: DIECKMANN‐SCHUPPERT Angela , BAUSE Ernst , SCHWARZ Ralph T.
Published: March 2005

O‐Glycosylation is the major form of protein glycosylation in human erythrocytes infected with the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum. This study compares aspects of O‐glycosylation in P. falciparum‐infected and uninfected erythrocytes.

Non‐labeled and metabolically glucosamine‐labeled O‐glycans were obtained from the protein fraction of infected or uninfected erythrocytes by β elimination. Additional label was introduced by reduction with sodium borohydride, or by the attachment of radioactive Gal to peripheral GlcNAc using galactosyltransferase. 2–4‐times more labeled O‐glycans were obtained from infected erythrocytes compared to the same number of uninfected ones, consistent with additional biosynthesis by the parasite. Our analysis of these O‐glycans showed no significant qualitative divergence between the O‐glycans of the infected and those of the uninfected red cell.

According to preliminary alditol analyses, the O‐glycans of P. falciparum‐infected red cells do not contain GalNAc at their reducing terminus. Moreover, GalNAc was not synthesized by P. falciparum from either Glc, Gal, GlcN or GalN. At least one O‐glycan found in P. falciparum‐infected erythrocytes contains GlcNAc at its reducing terminus.

Gel‐filtration results had suggested the presence of O‐GlcNAc on proteins in the infected erythrocyte. Probing with a synthetic pentapeptide, we could show that P. falciparum expresses its own O‐GlcNAc transferase during intracrythrocytic development. Using this peptide, the enzyme was characterized to some degree. The localization and function of O‐GlcNAc in P. falciparum remains to be elucidated.
Variation of the glycosylation of human pancreatic bile‐salt‐dependent lipase
Authors: MAS Eric , ABOUAKIL Nezha , ROUDANI Samira , FRANC Jean‐Louis , MONTREUIL Jean , LOMBARDO Dominique
Published: March 2005

Glycoprotiens of human pancreatic juice were characterized by means of lectins after electrophoresis and electrotransfer to nitrocellulose membranes. For the detected glycoproteins, only a 100‐kDa glycoprotein varied in the pancreatic juice from a normal patient (i.e, without any pancreatic disorder) compared to the pancreatic juice from a patient suffering from chronic pancreatitis. This protein, which is the only protein in human pancreatic juice which is O‐glycosylated and N‐glycosylated, was identified as the bile‐salt‐dependent lipase. Among the glycosylated proteins present in human pancreatic juice, only the glycosylation of bile‐salt‐dependent lipase differs between individuals. The enzyme was isolated either from normal or pathological human pancreatic juices. The purified variants have an identical molecular mass and amino‐acid compositon. As suspected from lectin affinity studies, the oligosaccharide composition differs between the variants. The structure of the N‐linked oligosaccharides of the variant from the pancreatic juice ofa normal donor correlated with complete processing and maturation of a complex‐type N‐glycan. Alteration of the maturation process can be detected for a bile‐salt‐dependent‐lipase variant from a patient suffering with chronic pancreatitis, since the carbohydrate composition is compatible with the predominance of hybrid or high‐mannose‐type structures. The amount of sugar involved in O‐glycosylation associated with the peanut agglutinin reactivity suggests the presence of 12–14 minimal Gal β1→3GalNAc→T/S O‐glycan structures which are sialylated and fucosylated. The amount of sugar involved in the O‐linked oligosaccharide structure appears to be unchanged in the variants isolated from the pathological pancreatic juice.
The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen
Authors: VILLALBA Mayte , BATANERO Eva , LÓPEZ‐OTÍN Carlos , SÁNCHEZ Luis M. , MONSALVE Rafael I. , GONZÁLEZ DE LA PEÑA Manuel A. , LAHOZ Carlos , RODRÍGUEZ Rosalía
Published: March 2005

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which has been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.
Application of an ectopic expression system for the selection of protein‐isoform‐specific antibodies
Authors: REINHARDT‐MAELICKE Sigrid , KURZ Sabine , SEWING Sabine , STOCKER Martin , PONGS Olaf
Published: March 2005

Monoclonal antibodies were raised against a fusion protein consisting of a fragment of 141 amino acids of the C‐terminal region of the rat brain voltage‐gated K+‐channel protein (RCK1) and the λLDN protein (fusion protein I). Selection of K+‐channel‐specific hybridoma cell lines was performed by means of an ELISA employing a fusion protein consisting of the K+‐channel‐specific peptide sequence and glutathione S‐transferase (fusion protein II). For final selection of RCK1 isoform‐specific antibodies, a panel of Xenopus oocytes was employed, each injected with cRNA coding for a specific RCK isoform (RCK 1, 2, 4 or 5). Several days after injection, cryosections of embedded oocytes were obtained and were employed in immunohistochemical analysis of antibody binding. Of five hybridoma supernatants from stable growing hybridoma cell lines, selected by the fusion‐protein ELISA, one monoclonal antibody (denoted K1C3) recognized exclusively the RCK1‐protein isoform, with the other four exhibiting different levels of cross‐reactivity with other K+‐channel isoforms, or with unknown protein(s) of non‐injected oocytes. The expression of the RCK1 protein in the postnatal brain was studied using, as fas as we are aware, the first example of the application of such isoform‐specific antibodies
Aberrant proteolysis of the β‐amyloid precursor protein in familial Alzheimer's disease lymphoblastoid cells
Authors: MATSUMOTO Akira , FUJIWARA Yoshisada
Published: March 2005

Lymphoblastoid cells from patients with early‐onset and late‐onset familial Alzheimer's disease showed increased expressions of β‐amyloid precursor mRNA and protein as well as interleukin‐1 and α1‐antichymotrypsin protein. Early‐onset and late‐onset familial Alzheimer's disease cells had greater production of 16‐kDa β‐amyloid C‐terminal preamyloid peptides than did normal cells. A pulse‐chase experiment indicated that aberrant processing of this peptide resulted in its abnormal accumulation. Furthermore, the peptide prepared from early‐onset familial Alzheimer's disease cells using formic acid could be separated into four discrete fragments. The N‐terminal amino acid sequencing of each fragment indicated that the 16‐kDa peptide was generated by cleavage, principally at the 30 amino acids N‐terminal to β‐amyloid. Both the enhanced synthesis and aberrant processing of the β‐amyloid precursor protein, therefore, are basic processes associated with familial Alzheimer's disease lymphoblastoid cells.
Cloning, sequencing and immunological characterization of the corrinoid‐containing subunit of the N5‐methyltetrahydromethanopterin: coenzyme‐M methyltransferase from Methanobacterium thermoautotrophicum
Authors: STUPPERICH Erhard , JUZA Andreas , HOPPERT Michael , MAYER Frank
Published: March 2005

A 3.5‐kb EcoRI fragment of the Methanobacterium thermoautotrophicum chromosome contains five open reading frames, mtrA to mtrE. The deduced N‐terminal amino acid sequence of mtrA is identical with 26 N‐terminal amino acids of a corrinoid‐containing membrane protein from Methanobacterium. Computer‐aided analyses of mtrA predicts 237 amino acids with a molecular mass of 25603 Da for its gene product. A hydropathy plot of this amino acid sequence indicates one hydrophobic helical conformation near the N‐terminus of the peptide which represents a tentative membrane‐spanning region. The main part of the protein, however, shows hydrophilic domains, suggesting a location outside the cytoplasmic membrane. These domains are probably accessible by monospecific polyclonal antibodies raised previously against the corrinoid‐containing membrane protein. The immunogold‐labeling technique revealed that the corrinoid‐dependent membrane protein was detectable at the cytoplasmic face of the membranes and of vesicle preparations. No significant identity of the deduced amino acid sequence was found with sequences of several corrinoid‐containing enzymes. In contrast to the hydrophilic gene product of mtrA, four other gene products from the gene cluster encode extremely hydrophobic proteins. The N‐terminal sequences of mtrC and mtrD are identical with two peptides of the N5‐methyltetrahydromethanopterin:coenzyme‐M methyltransferase complex from Methanobacterium, indicating that the mtr genes encode this membrane protein.
Activation of porcine pepsinogen A
Authors: NIELSEN F. Stausholm , FOLTMANN Bent
Published: March 2005

Reaction products formed during activation of porcine pepsinogen A at pH 2 were characterized by native agar‐gel electrophoresis and by denaturing SDS/PAGE. The results revealed the presence of non‐covalent intermediates between prosegment peptides and pepsin. The complexes Leu1p–Leu44p/pepsin and Leu1p–Leu16p/pepsin were isolated (the prosegment residues are characterized by the suffix p; numbering of residues starts again from the N‐terminus of pepsin). Relative to mature pepsin, the inherent milk‐clotting activities of the intermediates were 3% and 18%, respectively. The intermediates were incubated at pH 8.5 for 20 min at 28°C and the residual proteolytic activities were tested at pH 2. The stabilities at pH 8.5 were between those of pepsinogen and pepsin, Leu1p‐Leu44p/pepsin being most stable. The implications of these findings for the conformational changes that occur during the activation of pepsinogen are discussed.
Synthesis of charybdotoxin and of two N‐terminal truncated analogues
Authors: VITA Claudio , BONTEMS Françoise , BOUET Françoise , TAUC Michel , POUJEOL Philippe , VATANPOUR Hossein , HARVEY Alan L. , MENEZ André , TOMA Flavio
Published: March 2005

Charybdotoxin and two N‐terminal truncated peptides, corresponding to the 2–37 and 7–37 sequences, were obtained by stepwise solid‐phase synthesis using Nα‐t‐butyloxycarbonyl and benzyl‐type side‐chain protection. While this strategy was generally useful, the S‐acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high‐pressure liquid chromatography with overall yields of 4.0–5.0%. Each protein was characterised chemically, structurally and functionally. 1H‐NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three‐dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca2+‐activated K+ channels and on rat brain synaptosomal voltage‐dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5‐oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1–6 segment reduced potency much more. We conclude that the N‐terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable α/β fold can be engineered with only 31 amino acid residues.
Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two‐dimensional NMR
Authors: ESPOSITO Gennaro , FOGOLARI Federico , VIGLINO Paolo , CATTARINUSSI Serge , DE MAGISTRIS Maria Teresa , CHIAPPINELLI Lorella , PESSI Antonello
Published: March 2005

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both α and ε linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form α‐helix.

The peptide DNVLDHLTGR, its N‐acetylated and C‐amidated analogue and a tetrabranched MAP based on the N‐acetylated peptide were prepared and studied by CD and two‐dimensional 1H‐NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in trifluoroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3–Thr8 in the uncapped molecule and encompassing also the N‐terminal region in the capped analogue. The mobile C‐terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single‐copy epitope in the four‐branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end‐blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to εNH was found in a much more extended conformation than the corresponding link to αNH.

These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.
Hydrogen formation from glycolate driven by reversed electron transport in membrane vesicles of a syntrophic glycolate‐oxidizing bacterium
Authors: FRIEDRICH Michael , SCHINK Bernhard
Published: March 2005

Oxidation of glycolate to 2 CO2 and 3 H2 (ΔG°′=+36 kJ/mol glycolate) by the proton‐reducing, glycolate‐fermenting partner bacterium of a syntrophic coculture (strain FlGlyM) depends on a low hydrogen partial pressure (pH2). The first reaction, glycolate oxidation to glyoxylate (E°′=–92 mV) with protons as electron acceptors (E°′=–414 mV), is in equilibrium only at a pH2 of 1 μPa which cannot be maintained by the syntrophic partner bacterium Methanospirillum hungatei; energy therefore needs to be spent to drive this reaction. Glycolate dehydrogenase activity (0.3–0.96 U · mg protein−1) was detected which reduced various artificial electron acceptors such as benzyl viologen, methylene blue, dichloroindophenol, K3[Fe(CN)6], and water‐soluble quinones. Fractionation of crude cell extract of the glycolate‐fermenting bacterium revealed that glycolate dehydrogenase, hydrogenase, and proton‐translocating ATPase were membrane‐bound. Menaquinones were found as potential electron carriers. Everted membrane vesicles of the glycolate‐fermenting bacterium catalyzed ATP‐dependent H2 formation from glycolate (30–307 nmol H2· min−1· mg protein−1). Protonophores, inhibitors of proton‐translocating ATPase, and the quinone analog antimycin A inhibited H2 formation from glycolate, indicating the involvement of proton‐motive force to drive the endergonic oxidation of glycolate to glyoxylate with concomitant H2 release. This is the first demonstration of a reversed electron transport in syntrophic interspecies hydrogen transfer.
Combined plasma‐desorption mass spectrometry and Edman degradation applied to simultaneous sequence determination of isoforms of structural proteins from the cuticle of Locusta migratoria
Authors: ANDREASEN Lea , HøJRUP Peter , ANDERSEN Svend O. , ROEPSTORFF Peter
Published: March 2005

The primary structures of two basic low‐molecular‐mass proteins, Lm‐67 and Lm‐70 from the pharate cuticle of the migratory locust, Locusta migratoria, were determined. The sequencing strategy was based on combined use of plasma–desorption mass spectrometry (PDMS) and automatic Edman degradation of the proteins and their enzymically derived peptides. The mass‐spectral data showed the presence of two proteins in each preparation. For protein preparation Lm‐67, this was indicated by the mass spectrum of the intact protein. For protein preparation Lm‐70, the presence of two variants only became evident by mass‐spectrometric analysis of the enzymically derived peptides. Both proteins show strong similarity to other exocuticular proteins from L. migratoria.
Calcium binding to sialic acids and its effect on the conformation of ependymins
Authors: GANSS Bernhard , HOFFMANN Werner
Published: March 2005

Soluble ependymins form the predominant protein constituents in the cerebrospinal fluid from many orders of teleost fish. Furthermore, these glycoproteins also exist in a bound form associated with the extracellular matrix. Ependymins are synthesized in meningeal fibroblasts. In goldfish, their synthesis is increased during the regeneration of the optic nerve and they share several characteristics with molecules involved in cell contact phenomena. In this study, we show by a calcium overlay technique that ependymins from goldfish and rainbow trout are able to bind 45Ca2+. However, nearly all of this Ca2+‐binding capacity is lost after digestion with sialidase. Furthermore, circular‐dichroism spectra from FPLC‐purified rainbow trout ependymins have been recorded in the presence and absence of Ca2+. Below 250 nm, the CD spectrum showed a characteristic minimum of ellipticity at 217 nm typical of β structures. This signal is independent of the Ca2+ concentration. In contrast, the complex signal at 250–310 nm mainly decreased with increasing Ca2+ concentration indicating changes in the environment of aromatic side chains.