bluuebunny/crossref_metadata_2025 huggingface dataset:
c‐fos Protooncogene Transcription can be Modulated by Oligonucleotide‐Mediated Formation of Triplex Structures in vitro
Authors: Lavrovsky Yan , Stoltz Robert A. , Vlassov Valentin V. , Abraham Nader G.
Published: August 2004

A homopurine · homopyrimidine sequence of the c‐fos promoter was chosen as a target for a triple helix oligonucleotide. Eight DNA oligonucleotides that ranged from 14 to 31 bp were shown to form a triple helix with three sequences within the c‐fos promoter region. Reactive derivatives of homopyrimidine oligonucleotides bearing the 5′‐or 3′‐terminal DNA alkylation aromatic 2‐chloroethylamino group were also synthesized. It was concluded, based on the physical properties of the DNA oligonucleotide complex, that the oligonucleotide forms a colinear triplex with the duplex binding sites. We investigated in detail, using electrophoretic mobility and footprinting protection, whether such oligonucleotide · DNA complexes are of benefit in designing high‐affinity probes for a natural DNA sequence in the mouse c‐fos gene. Our results demonstrate that four different DNA targets within the c‐fos promoter region can form triplex structures with synthetic oligonucleotides in a sequence‐specific manner. Moreover, in vitro modifications of the retinoblastomagene‐product‐binding site of the c‐fos promoter at position–83 in front of the cAMP/cAMP‐responsive element binding site and fos ‐binding site 3/activator‐protein‐2‐like (FBS3/AP‐2‐like) site at position–431 by triple helix forming oligonucleotides cause dramatic suppression of fos ‐chloramphenicol acetyltransferase activity in endothelial cells. These results provide a basis for the development of a specific oligonucleotide target forming triplex‐DNA complex, and emphasize the importance of a target forming triplex as a basis for control of gene expression and cell proliferation.
Structural Studies of the O‐Specific Polysaccharide of Hafnia alvei Strain 1209 Lipopolysaccharide
Authors: Niedziela Tomasz , Petersson Carl , Helander Anne , Jachymek Wojciech , Kenne Lennart , Lugowski Czeslaw
Published: August 2004

The structure of the O‐specific side chains of the Hafnia alvei strain 1209 lipopolysaccharide has been investigated. Methylation analysis and 1H‐NMR and 13C‐NMR spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating units that have the following structure:
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The relative intensity of the signals from the terminal repeating unit in the 1H‐NMR spectrum, the amount of 2,3,6‐tri‐O ‐methylgalactose in the methylation analysis, and the matrix‐assisted laser‐desorption ionisation time‐of‐flight (MALDI‐TOF) mass spectrum of the O‐polysaccharide indicated that the structure is also the biological repeating unit and that the O‐chains mainly consisted of 8–11 repeating units and, on average, ten repeating units.
Cell‐free Conversion of a Ubiquitous Nuclear Protein into a Killer‐Cell‐Specific form that Binds to the Nf‐P Enhancer Element of the Mouse Perforin Gene
Authors: Horta Maria de Fátima , Fu Kok‐Chin , Koizumi Hirotaka , Young John Ding‐E , Liu Chau‐Ching
Published: August 2004

Two nuclear factors, designated NF‐P1 and NF‐P2, have been shown to bind to an enhancer 9‐base motif (5′‐ACAGGAAGT‐3′, NF‐P motif) present within the 5′‐flanking region of the mouse perforin gene. Our previous studies have shown that, although NF‐P1 and NF‐P2 differ in cell‐type distribution and molecular mass, with NF‐P2 being killer‐cell‐specific and smaller, the two factors appear to share common DNA‐binding subunit(s). We have postulated that the biochemical event involved in the induction of NF‐P2 could be the dissociation of a non‐DNA‐binding subunit from NF‐P1, rendering the newly formed NF‐P2 transcriptionally active. By using a cell‐free system in the present study, we have demonstrated that a variety of chemical agents capable of denaturing or dissociating protein complexes, including guanidinium/HCl, detergents (SDS plus Nonidet P‐40) and high‐salt solutions, could convert NF‐P1 into NF‐P2. Unlike in intact cells, where induction of NF‐P2 is restricted to killer lymphocytes, this conversion occurred in nuclear extracts derived from both cytotoxic lymphocytes and non‐cytotoxic cells. Although the mechanism that restricts the induction of NF‐P2 to killer lymphocytes in vivo remains unresolved, these results support the hypothetical ‘dissociation’ model for the generation of NF‐P2. The results also imply that the absence of perforin expression in non‐cytotoxic cells in vivo may be due to the suppression of the induction of the killer‐cell‐specific trans‐acting factor NF‐P2.
Interaction of the La (SS‐B) Autoantigen with Small Ribosomal Subunits
Authors: Peek Ron , Pruijn Ger J. M. , Van Venrooij Walther J.
Published: August 2004

The La (SS‐B) autoantigen is an evolutionarily conserved 47‐kDa protein which binds to nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) messenger RNAs. Here, we show that La in the cytoplasm is associated with a subset of small ribosomal subunits, possibly by direct association with 18S ribosomal RNA. This association is likely to be related to the putative role of this protein in translation regulation.
The Aspergillus nidulans Penicillin‐Biosynthesis Gene aat (penDE) is Controlled by a CCAAT‐Containing DNA Element
Authors: Litzka Olivier , Then Bergh Katharina , Brakhage Axel A.
Published: August 2004

Analysis of the promoter of the penicillin biosynthesis aat (penDE) gene of Aspergillus nidulans using band‐shift assays led to the identification of a CCAAT‐containing DNA element which was specifically bound by a protein (complex). The identified DNA element was localised about 250 bp upstream of the transcriptional‐start sites of aat. Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat‐lacZ gene fusion. The identified binding site thus was functional in vivo and positively influenced aat expression. Partial purification of the CCAAT binding protein and cross‐competition experiments provided evidence that the binding protein is identical to the identified putative penicillin‐regulatory protein PENR1, binding to the CCAAT element in the bidirectional in‐tergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENR1 seems to be involved in the regulation of all three penicillin‐biosynthesis genes. Cross‐competition experiments demonstrated that the promoter region of the corresponding aat (penDE) gene of Penicillium chrysogenum was capable to dilute the shift of the A. nidulans probe with PENR1, suggesting the presence of a similar regulatory mechanism in this fungus. Taken together with previous data, CCAAT‐containing DNA elements thus seem to represent major cis‐acting sites in the promoters of β‐lactam‐biosynthesis genes.
The Copper‐topaquinone‐phenylhydrazine‐adduct Geometry in Escherichia coli Amine Oxidase Derivatized with Phenylhydrazines Substituted with Trifluoromethyl Groups, as Determined with 19F‐NMR Relaxation Measurements
Authors: Steinebach Vincent , De Jong Govardus A. H. , Wijmenga Sybren S. , De Vries Simon , Duine Johannis A.
Published: August 2004

The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazines substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F‐NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line‐broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon‐Bloembergen equation for the distance‐dependent contribution of Cu(II) to the transversal‐relaxation time of the F resonance. Assuming that the F3C‐phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry‐related counterpart above or below the topaquinone‐phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 ± 0.06 nm from the C2‐O, 1.30 ± 0.04 nm from the C4‐O, and 1.26 ± 0.04 nm from the C5‐N). However, one of the configurations yields very similar distances between the Cu and the C2‐O and C4‐O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F‐NMR data obtained for porcine‐plasma amine oxidase [Williams, T. J. & Falk, M. C. (1986) J. Biol. Chem. 261, 15949–15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.
Purification and cDNA Cloning of the Alcohol Dehydrogenase of the Flesh Fly Sarcophaga peregrine
Authors: Horio Tomohiro , Kubo Takeo , Natori Shunji
Published: August 2004

We purified to homogeneity two proteins with molecular masses of 25 kDa from the fat body of the Sarcophaga larva. One was alcohol dehydrogenase (ADH) and the other was a 25‐kDa protein of which the genomic DNA had been cloned. We isolated the cDNA for ADH and determined its amino acid sequence. Amino acid sequence identity between ADH and the 25‐kDa protein was 40%, indicating that they are structurally related proteins. The amount of ADH in Sarcophaga was almost constant through the larval stage to the adult stage, but the 25‐kDa protein was detected only within a restricted period between the final larval instar and the early pupal stage.
Proteases from Trypanosoma brucei brucei
Authors: Troeberg Linda , Pike Robert N. , Morty Rory E. , Berry Ronald K. , Coetzer Theresa H. T. , Lonsdale‐Eccles John D.
Published: August 2004

African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain‐Tb) and a serine oligopeptidase (which we call oligopeptidase‐Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules.

The enzymes differed in various respects. Firstly, purified trypanopain‐Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase‐Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor.

The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and α2‐macroglobulin effectively inhibited trypanopain‐Tb, with the Ki values for cystatin C and low‐molecular‐mass kininogen (≈10−11 M) predicting that trypanopain‐Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase‐Tb was not inhibited by serpins or α2‐macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome‐infected rats displayed no trypanopain‐Tb‐like activity, but exhibited high oligopeptidase‐Tb‐like activity. Thus, while trypanopain‐Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase‐Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.
Substitution of Fifty Four Homologue (Ffh) in Escherichia coli with the Mammalian 54–kDa Protein of Signal‐Recognition Particle
Authors: Patel Sarju , Austen Brian M.
Published: August 2004

The fifty four homologue (Ffh) of Escherichia coli promotes the translocation of a subset of periplasmic, membrane and secreted proteins across the cytoplasmic membrane. The ffh gene product is essential for cell viability and efficient protein export. Here we show that the mammalian homologue signal‐recognition particle (SRP) 54 kDa is not able to suppress the translocation defect in an Ffh conditional mutant Wam 113 [Phillips, G. J. & Silhavy, T. J. (1992) Nature 359, 744–746], The expression of SRP 54 kDa, which is increased when Ffh is suppressed in the Wam 113 strain, causes a pleiotropic defect characterised by cell elongation, and increased accumulation of precursor proteins. The accumulation of precursors of outer membrane protein A (Omp A) and maltose‐binding protein (MBP), Sec‐B dependent pre‐proteins, was less than the Ffh‐dependent proteins ribose‐binding protein (RBP) and β‐lactamase. Sec B expression was suppressed by Ffh expression. The recombinant SRP 54 kDa, which forms a ribonucleoprotein complex in E. coli, was shown to bind to precursor proteins, but is unable to interact with the filamentous temperature‐sensitive Y (Fts Y) membrane receptor of the translocation machinery.
Characterization of the Interaction Between Annexin I and Profilin
Authors: Alvarez‐Martinez Maria‐Teresa , Mani Jean‐Claude , Porte Françoise , Faivre‐Sarrailh Catherine , Liautard Jean Pierre , Widada Joannes Sri
Published: August 2004

Annexin I belongs to a family of calcium‐dependent phospholipid‐binding and membrane‐binding proteins. Although many of the biochemical properties and the three‐dimensional structure of this protein are known, its true physiological roles have yet to be thoroughly defined. Its putative functions include participation in the regulation of actin microfilaments dynamics, proposed after the discovery of an interaction with actin. In accordance with this hypothesis, we found that annexin I can also interact with profilin. We used different methods, overlay and surface plasmon resonance (BIAcore), to measure the parameters of the association equilibrium, i.e. kon, koff and Kd. The affinity of annexin I for profilin was between 107 M and 108 M. High concentrations of KCl did not prevent the interaction, although a slight decrease in affinity was observed. Calcium, a modulator of annexin I functions interfered only marginally with the association, in a manner comparable to magnesium. Proteins or compounds known to interact with annexin I or profilin were found to inhibit the annexin‐I–profilin interaction when added in the reaction medium. Recombinant profilin exhibited a slightly lower affinity than natural platelet protein when measured with BIAcore. Due to the submembrane localisation of annexin I and the regulatory activity of profilin on the cytoskeleton, an interaction between annexin I and profilin may therefore be implicated in the regulation of some cellular functions, particularly those governing membrane‐cytoskeleton dynamic organization.
Experimental Procedure for a Hydrogen Peroxide Assay Based on the Peroxidase‐Oxidase Reaction
Authors: Demmano Gustave , Selegny Eric , Vincent Jean‐Claude
Published: August 2004

A low‐cost assay method that is able to measure H2O2 concentrations as low as the nano‐molar range is described. The assay solution contains NADH, horseradish peroxidase, and superoxide dismutase at pH 7.5. After the addition of the sample, the decrease in NADH concentration measured by spectrophotometry is proportional to the H2O2 concentration. Because of superoxide dismutation, a high amplification factor defined as moles NADH oxidised per mole H2O2 added is obtained, which allows the sensitivity limit of the method to be greatly improved. We have established the conditions under which the amplification factor can be stabilised at a high level: the best compromise is to increase both the horseradish peroxidase and superoxide dismutase concentrations. Finally, we have also shown that coupled to specific oxidases, our assay method is suitable for measuring very low concentrations of biochemicals that can be oxidized by oxygen with H2O2 production.
The Oligomeric State of 46‐kDa Mannose 6‐Phosphate Receptor does not Change Upon Intracellular Recycling and Binding of Ligands
Authors: Punnonen Eeva‐Liisa , Wilke Tanja , Von Figura Kurt , Hille‐Rehfeld Annette
Published: August 2004

46‐kDa mannose‐6‐phosphate receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent‐solubilized 46‐kDa mannose 6‐phosphate receptor is regulated by the presence of ligands, pH and receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47–54). To find out whether the intracellular recycling of 46‐kDa mannose 6‐phosphate receptor is accompanied by changes in its quaternary structure, we have performed chemical cross‐linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46‐kDa mannose 6‐phosphate receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20 % of total endogenous 46‐kDa mannose 6‐phosphate receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300‐kDa mannose 6‐phosphate receptor), nor upon depletion of adaptors from clathrin‐coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46‐kDa mannose 6‐phosphate receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46‐kDa mannose 6‐phosphate receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46‐kDa mannose 6‐phosphate receptor in situ, our data suggest that it represents the main physiologically active form of the receptor, and therefore present indirect evidence that binding of ligands to 46‐kDa mannose 6‐phosphate receptor is probably regulated by conformational changes of receptor or ligand rather than by changes in the quaternary structure.
The Complete cDNA Sequence of Laminin α4 and Its Relationship to the Other Human Laminin α Chains
Authors: Richards Allan , Al‐Imara Lamya , Pope F. Michael
Published: August 2004

We previously localised the gene (LAMA4) encoding a novel laminin α4 chain to chromosome 6q21. In this study, we describe the complete coding sequence and compare the protein with the other three known human laminin α chains. Although closely linked to LAMA2, the LAMA4 product most closely resembles laminin α3, a constituent of laminin 5. Like laminin α3A, the α4 chain is a truncated version of the α1 and α2 chains, with a much reduced short arm. While the α4 molecule is most similar to α3, it shares some features of the C‐terminal domains G4 and G5 in common with α2. Unlike the LAMA3 gene, LAMA4 appears to encode only a single transcript, as determined by 5′ rapid amplification of cDNA ends. The cDNA sequence encodes 1816 amino acids, which include a 24‐residue signal peptide. The gene is expressed in skin, placenta, heart, lung, skeletal muscle, and pancreas. We have also shown that the mRNA can be readily reverse transcribed and amplified from cultured dermal fibroblasts.
Expression in Escherichia coli of Sin a 1, the Major Allergen from Mustard
Authors: De La Peña Manuel A. González , Monsalve Rafael I. , Batanero Eva , Villalba Mayte , Rodríguez Rosalía
Published: August 2004

Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family. It is composed of two disulfide‐bonded polypeptide chains. The cloning of this allergen has been carried out by means of the polymerase chain reaction using non‐degenerate oligonucleotides encoding the N‐terminal and C‐terminal regions of the mature protein as primers. Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment. The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein. One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S ‐transferase from Schistosoma japonicum. The resulting chimeric protein was purified by affinity chromatography on a glutathione–Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen. The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard‐sensitive human sera. In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen.
Alternative mRNA Splicing of the Novel GTPase Rab28 Generates Isoforms with Different C‐Termini
Authors: Brauers Andreas , Schürmann Annette , Massmann Silke , Mühl‐zürbes Petra , Becker Walter , Kainulainen Heikki , Lie Chichung , Joost Hans‐Georg
Published: August 2004

A novel ras‐related gene (rab 28) was identified by a PCR‐based cloning approach and subsequent screening of rat fat cell and brain cDNA libraries. The deduced amino acid sequence of the cDNA is distantly related with members of the Rab family (31–33% sequence identity, mainly restricted to the six GTP‐binding motifs). Cloning of the human homologue of Rab28 by a PCR‐based approach revealed the existence of two isoforms (hRab28S, hRab28L) which differ only by a 95–bp insertion within the coding region. This insertion generates an alternative sequence of the 30 C‐terminal amino acids of the protein. Both C‐termini of the human homologues comprise farnesylation motifs, but differ strikingly in a stretch of 13 amino acids. By PCR, mRNA of hRab28S was detected in most tissues investigated (cortex, liver, kidney, skeletal muscle, adipose tissue, testis and urothelium), whereas hRab28L was predominant in testis. Recombinant Rab28 proteins showed specific binding of radiolabeled guanosine 5′‐O‐[γ‐thio]triphosphate and rapidly hydrolysed [α‐32P]GTP; there was no difference in the GTP binding characteristics of the two isoforms hRab28S and hRab28L. It is suggested that the isoforms are derived from the same gene by alternative mRNA splicing, and that their functions differ in a parameter unrelated to its basic role as a GTPase.
A Selenium‐Containing Phospholipid‐Hydroperoxide Glutathione Peroxidase in Schistosoma mansoni
Authors: Maiorino Matilde , Roche Catherine , Kiess Michael , Koenig Kerstin , Gawlik Dieter , Matthes Monika , Naldini Emanuela , Pierce Raymond , Flohé Leopold
Published: August 2004

The 1000000×g supernatant of the parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzyme phospholipid‐hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second‐order rate constants for the oxidation of the ground‐state enzyme (k+1) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene hydroperoxide or H2O2, and quantitatively similar to those of mammalian phospholipid‐hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium‐containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149–152]. S. mansoni thus contains a selenoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid‐hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.
Human N‐Acetylglucosaminyltransferase III Gene is Transcribed from Multiple Promoters
Authors: Koyama Nobuto , Miyoshi Eiji , Ihara Yoshito , Kang Rujun , Nishikawa Atsushi , Taniguchi Naoyuki
Published: August 2004

We have isolated cDNA clones for the human N‐acetylglucosaminyltransferase III (GlcNAc‐transferase III) gene. Two of them, H15 and H20, contain 5′ non‐coding regions that are totally different from each other except for 8 bp adjacent to the putative initiation codon. Analysis of one of the genomic cosmid clones containing the GlcNAc–transferase III coding region, Hug3, revealed that the 5′ non‐coding regions of H15 and H20 contain two and one exons, respectively, in addition to the exon containing the coding region (exon 1). These have arisen as the result of alternative splicing. The transcription‐initiation sites were determined by primer‐extension analysis and 5′‐rapid amplification of cDNA ends (RACE). Both H15‐specific and H20‐specific primers gave cDNAs longer than those expected from the lengths of H15 and H20, and a primer complementary to the region around the intron/exon junction near the putative initiation codon also gave distinct signals. Promoter activities of the 5′‐flanking regions of H15, H20 and exon 1 were measured in a human hepatoblastoma cell line, HuH‐6 cells by luciferase assays. The 5′‐flanking region of exon 1 was the most active, whilst that of H15 was several times less active, and that of H20 was inactive. Our study suggests that multiple promoters of the GlcNAc‐transferase III gene contribute to the complex regulation of this gene.
Thermal Unfolding and Proteolytic Susceptibility of Ribonuclease A
Authors: Arnold Ulrich , Rücknagel Karl Peter , Schierhorn Angelika , Ulbrich‐Hofmann Renate
Published: August 2004

With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20–65°C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electropho‐retic bands, partial N‐terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix‐assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31‐Ser32 and Arg33‐Asn34 for trypsin and Asn34‐Leu35 and Thr45‐Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent β‐structure containing Thr45‐Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.
Kinetics and Energetics of Trehalose Transport in Saccharomyces cerevisiae
Authors: Stambuk Boris U. , De Araujo Pedro S. , Panek Anita D. , Serrano Ramon
Published: August 2004

Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H+ depletion to trehalose uptake by yeast cells was approximately 1:1, which indicates the existence of a trehalose‐H+ symporter in these cells. The optimum pH for this active H+‐trehalose symport was 5.0, and both the Km and the Vmax were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high‐affinity H+‐trehalose symporter (Km= 4 mM), and a low‐affinity transport activity (Km >100 mM) that could be a facilitated diffusion process. The high‐affinity H+‐trehalose symporter was repressed by glucose, whereas the low‐affinity uptake was constitutively expressed in S. cerevisiae.
Gene Structure of the Murine Calcium Channel β3 Subunit, cDNA and Characterization of Alternative Splicing and Transcription Products
Authors: Murakami Manabu , Wissenbach Ulrich , Flockerzi Veil
Published: August 2004

The β3 subunit of high‐voltage‐gated calcium channels is a peripheral membrane protein that copurifies with neural N‐type calcium channels. Murine genomic clones containing the full coding sequence of β3 were isolated and the exons were mapped and sequenced. The murine calcium channel β3 subunit is encoded by a unique gene composed of 13 translated exons that spread over ≈8 kb of genomic sequence. Alternatively spliced transcripts of the β3 gene were identified and characterized. The primary structure of β3 is highly conserved between the murine, human, rabbit and rat proteins (98% identity). The intron placement within that primary structure correlates with the previously postulated exon positions in transcripts encoding the members of the calcium channel β subunit family and confirm a close evolutionary relationship of the β3, β1, β2 and β4 subunit genes.
Polymerization of 5,6‐Dihydroxyindole‐2‐Carboxylic Acid to Melanin by the Pmel 17/Silver Locus Protein
Authors: Chakraborty Ashok K. , Platt James T. , Kim Kack K. , Se Kwon Byoung , Bennett Dorothy C. , Pawelek John M.
Published: August 2004

Recent advances in melanogenesis have focused on the role of dihydroxyindole‐2‐carboxylic acid [(HO)2IndCOOH]. For example, it has been shown that formation of (HO)2IndCOOH from dopachrome is catalyzed by dopachrome tautomerase, that the melanogenic protein tyrosinase‐related protein (TRP)‐1 can oxidize (HO)2IndCOOH to its indole quinone, that (HO)2IndCOOH‐melanms can be synthesized chemically, that mammalian melanins are naturally rich in (HO)2IndCOOH subunits, and that (HO)2IndCOOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)2IndCOOH and other precursors become incorporated into melanins in vivo. Accordingly, an activity was partially purified that catalyzed melanin formation with (HO)2IndCOOH as a substrate. Analyses of the (HO)2IndCOOH polymerization factor from Cloudman melanoma cells revealed the following: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N‐acetyl glucosamine; it was located predominantly in the melanosomal fraction of cell homogenates; the activity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)2IndCOOH polymerization reaction was inhibited by superoxide dismutase. In addition, the activity was found with the mouse pmel 17/silver locus protein immunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice compared to extracts from Si/Si melanocytes. In summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide‐dependent polymerization of (HO)2IndCOOH to melanin in vitro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus.
Identification of a Cysteine Protease Closely Related to Interleukin‐1β‐Converting Enzyme
Authors: Faucheu Chi , Blanchet Anne‐Marie , Collard‐Dutilleul Véronique , Lalanne Jean‐Louis , Diu‐Hercend Anita
Published: August 2004

The present study describes the identification and molecular cloning of a new member of the interleukin‐1β‐converting enzyme (ICE) family denoted transcript Y (TY). TY is very closely related to both ICE (51% amino acid identity) and a protein named transcript X (TX) (75% amino acid identity) that we recently identified [Faucheu, C., Diu, A., Chan, A. W. E., Blanchet, A.‐M., Miossec, C., Hervé, P., Collard‐Dutilleul, V., Gu, Y., Aldape, R., Lippke, J., Rocher, C., Su, M. S.‐S., Livingston, D. J., Hercend, T. & Lalanne, J.‐L. (1995) EMBO J. 14, 1914–1922]. The amino acids that are implicated in both the ICE catalytic site and in the P1 aspartate‐binding pocket are conserved in TY. Within the ICE gene family, TY belongs to a subfamily of proteins closely related to the prototype ICE protein. Using transfection experiments into mammalian cells, we demonstrate that TY has protease activity on its own precursor and that this activity is dependent on the presence of a cysteine residue at position 245. However, despite the close similarity between TY and ICE active sites, TY fails to process the interleukin‐1β precursor. In addition, as already observed for ICE and TX, TY is able to induce apoptosis when overexpressed in COS cells. TY therefore represents a new member of the growing family of apoptosis‐inducing ICE‐related cysteine proteases.
Different Apoptotic Pathways Mediated by Fas and the Tumor‐Necrosis‐Factor Receptor
Authors: Enari Masato , Hug Hubert , Hayakawa Makio , Ito Fumiaki , Nishimura Yoshifumi , Nagata Shigekazu
Published: August 2004

Fas is a cell‐surface receptor that belongs to the tumor‐necrosis factor (TNF)/nerve growth factor receptor family. Fas can transduce an apoptotic signal through the death domain in the cytoplasmic region, which has similarity with the corresponding region of the TNF type‐I receptor. Here, we expressed human Fas in mouse L929 cells or its subline (C12), which express extremely low levels of cytosolic phospholipase A2 (cPLA2). L929 cells were sensitive to the cytotoxic activity of TNF, while C12 cells were resistant. Cross‐linking of human Fas with anti‐(human Fas antibody) Ig killed both L929 transformants and C12 transformants expressing human Fas. Various inhibitors of the arachidonate metabolism significantly inhibited the TNF‐induced cytotoxicity in L929 cells, but they did not have any effect on Fas‐mediated apoptosis. These results indicated that cPLA, is required for TNF‐induced apoptosis, whereas it is dispensable for Fas‐mediated apoptosis, and suggested that the TNF receptor and Fas use different signaling pathways for apoptosis.
A Receptor for the Import of Proteins into Human Mitochondria
Authors: Hanson Brendon , Nuttall Stewart , Hoogenraad Nicholas
Published: August 2004

We have characterised a 16.3‐kDa human protein that functions as a receptor for the import of preproteins into mitochondria. Based on amino acid sequence alignments, the protein (hMas20p) is 41% similar lo Mas20p (20‐kDa mitochondrial assembly protein) from yeast Saccharomyces cerevisiae and 38% similar to MOM19 (19‐kDa mitochondrial outer‐membrane protein) from Neurospora Crassa. hMas20p has a putative N‐terminal transmembrane sequence of 29 amino acids and an acidic C‐terminus. A 13‐kDa fragment [des‐(1–29)‐hMas20p], which lacks the 29‐amino‐acid putative N‐terminal transmembrane domain, is soluble when expressed in Escherichia coli Antibodies produced against this domain cross‐reacted with a protein or 16 kDa in outer membranes or mitochondria from rat liver and inhibited import of protein into isolated mitochondria from rat liver. In addition, the recombinant soluble domain folds into a functional structure as it competes with hMas20p on the mitochondrial surface for precursor binding, confirming the functional role of hMas20p in the import of precursor into mitochondria.
Maturation of Human Intestinal Lactase‐Phlorizin Hydrolase
Authors: Jacob Ralf , Radebach Ilona , Wüthrich Marcel , Grünberg Jürgen , Sterchi Erwin E. , Naim Hassan Y.
Published: August 2004

Human lactase‐phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine. is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH (LPHβ) by a trypsin‐like protease (Naim et al., 1987, 1991). Arg868‐Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site‐directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHβ species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N‐terminal end of brush border LPH at Lys851‐Arg852 and Arg830‐Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868‐Ala869 represents the final cleavage site that generates LPHβfinal. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868‐Ala869, to generate LPHinitial; LPHβinitial is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868‐Ala869 to mature brush border LPH (LPHβfinal).
The Human Cysteine‐Rich Secretory Protein (CRISP) Family
Authors: Krätzschmar Jörn , Haendler Bernard , Eberspaecher Uwe , Roosterman Dirk , Donner Peter , Schleuning Wolf‐Dieter
Published: August 2004

We report the isolation and characterisation of cDNAs encoding three different, human members of the cysteine‐rich secretory protein (CRISP) family. The novel CRISP‐I exists in five cDNA subtypes differing by the presence or absence of a stretch coding for a C‐terminal cysteine‐rich domain so far found in all members of the family, and by the length of their 3‐untranslated region. CRISP‐2 cDNA corresponds to the previously described TPXI form, with so far unreported 5′‐untranslated sequence heterogeneities while CRISP‐3 cDNA codes for a new, unique protein. Northern blot analysis of various human organs indicates that CRISP‐1 transcripts are epididymis‐specific whereas CRISP‐2/TPXI transcripts are detected mainly in the testis and also in the epididymis. CRISP‐3 transcripts are more widely distributed and found predominantly in the salivary gland, pancreas and prostate, and in less abundance in the epididymis, ovary, thymus and colon. A protein reacting with an anti‐mouse CRISP‐1 antibody was isolated from human epididymal extracts and N‐terminal sequencing revealed that it corresponded to the CRISP‐1 cDNA we have isolated. In contrast to findings on its rat counterpart epididymal protein DE/acidic epididymal glycoprotein (AEG), no significant association of CRISP‐1 with human spermatozoa was observed.
Social capital and its relevance to the Japanese‐model welfare society
Authors: Chan Raymond K H , Cheung Chau Kiu , Peng Ito
Published: August 2004

Current debates and initiatives relating to the welfare regime in Japan focus on the contributions of informal and community networks. In this article, we adopt the concept of social capital, which is assessed according to three categories – structural social capital, experiential individual social capital and anticipatory individual social capital – in order to evaluate the assumptions and strengths of community in Japan. The findings are based on a small‐scale survey conducted in the Greater Kobe area in 2002. The study revealed that the level of structural social capital is ‘average’ and the level of experiential individual social capital is ‘rather low’. However, the anticipatory individual social capital, which is the expectation of future assistance whether conditional or unconditional, is higher than the experiential individual social capital. The findings suggest that, in Japan, people's belief that they will receive assistance in the future has a significant impact on their level of achievement. Such findings may help us understand the nature of the welfare regime in Japan: it depends on a sense of general trust, which effectively supports the informal groups and community networks that provide assistance to their members.
PARALLELIZATION OF MONTE CARLO SIMULATIONS AND SUBMISSION TO A GRID ENVIRONMENT
Authors: MAIGNE LYDIA , HILL DAVID , CALVAT PASCAL , BRETON VINCENT , REUILLON ROMAIN , LAZARO DELPHINE , LEGRE YANNICK , DONNARIEIX DENISE
Published: August 2004

Monte Carlo simulations are increasingly used in medical physics. In scintigraphic imaging these simulations are used to model imaging systems and to develop and assess tomographic reconstruction algorithms and correction methods for improved image quantization. In radiotherapy-brachytherapy the goal is to evaluate accurately the dosimetry in complex phantoms and at interfaces of tissue, where analytic calculations have shown some limits. The main drawback of Monte Carlo simulations is their high computing time. The aim of our research is to reduce the computing time by parallelizing a simulation on geographically distributed processors. The method is based on the parallelization of the Random Number Generator (RNG) used in Monte Carlo simulations. The long serial of numbers used by the sequential simulation is split. Once the partitioning is done, a software application allows the user to generate automatically the files describing each simulation part. Finally, another software executes them on the DataGrid testbed using an API. All these steps have been made transparent for the user by providing a web page asking the user for all the parameters necessary to launch the simulation and retrieve results. Different tests have been done in order to show first, the reliability of the physical results obtained by concatenation of parallelized output data and secondly the time gained for jobs execution.
A CLIENT-SERVER PROTOTYPE FOR GRID-ENABLING APPLICATION TEMPLATE DESIGN
Authors: GREEN MARK L. , MILLER RUSS
Published: August 2004

A computational and data grid was developed at the Center for Computational Research in Buffalo, New York, in order to provide a platform to support scientific and engineering applications across a variety of computer and storage systems. This proof-of-concept grid has been deployed using a critical scientific application in the field of structural biology. The design and functionality of the prototype grid is described, along with plans for a production level grid system based on Globus.
The Announcement Effects of Restricted Open Market Share Repurchases: Experience from Taiwan
Authors: Chen Miawjane , Chen Chao-Liang , Cheng Wan-Hsiu
Published: August 2004

In this paper we empirically examine the effects of 451 restricted share repurchase announcements in Taiwan. Their 3-day cumulative abnormal returns are all significantly positive for different purposes and Tobin's qs. However, there is no significant difference in abnormal returns for different repurchasing purposes. This indicates that mandating a purpose is not really an effective tool for limiting managerial choice. Moreover, when the related variables are controlled, the other empirical results we conducted indicate that, at least in Taiwan, the traditional signaling hypothesis and the free cash flow hypothesis can function simultaneously to explain the effects of the restricted repurchase announcements.
Are Shocks Asymmetric to Volatility of Chinese Stock Markets?
Authors: Huang Wei-Chiao , Zhu Yuanlei
Published: August 2004

This paper uses ARCH models to examine if there is a leverage effect and also to test if A- and B-share holdings have different risks in Chinese stock markets before and after B-share markets open to domestic investors in February 2001. The empirical results suggest that leverage effect was not present and shocks have symmetric impact on the volatility of Chinese B-share stock returns in both periods and A-share returns in Period I. Thus GARCH model would be a better model to fit the Chinese B-share stock returns than EGARCH or GJR-GARCH model. But EGARCH or GJR-GARCH model fits recent (Period II) A-share markets data better than GARCH model. Another finding of this paper is that holding A- or B-share bears different risk in returns in the two Chinese markets. Furthermore, news or shocks have a larger impact on volatility of B-share returns in Period I than in Period II.
Modulating Angiogenesis
Authors: Sengupta Shiladitya , Toh Sue-Anne , Sellers Lynda A. , Skepper Jeremy N. , Koolwijk Pieter , Leung Hi Wun , Yeung Hin-Wing , Wong Ricky N.S. , Sasisekharan Ram , Fan Tai-Ping D.
Published: August 2004

Background—

Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis.

Methods and Results—

A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct “sterol ginsenoside” fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase→Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells.

Conclusions—

The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing.