bluuebunny/crossref_metadata_2025 huggingface dataset:
Introducing Wilson disease mutations into the zinc‐transporting P‐type ATPase of Escherichia coli
Authors: Okkeri Juha , Bencomo Eija , Pietilä Marja , Haltia Tuomas
Published: March 2003

ZntA, a bacterial zinc‐transporting P‐type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C‐terminal part of the phosphorylation domain in the so‐called hinge motif conserved in all P‐type ATPases. P401L resides near the N‐terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase‐specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0–37% of the wild‐type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and Pi. Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by Pi is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild‐type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild‐type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.
Thiol‐modifying inhibitors for understanding squalene cyclase function
Authors: Milla Paola , Lenhart Alexander , Grosa Giorgio , Viola Franca , Weihofen Wilhelm A. , Schulz Georg E. , Balliano Gianni
Published: March 2003

The function of squalene‐hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by site‐directed mutagenesis, with different thiol‐reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3‐carboxy‐4‐nitrophenyl‐dithio‐1,1′,2‐trisnorsqualene (CNDT‐squalene) showed that the cysteine residue at the channel constriction was covalently modified by the squalene‐like inhibitor. Time‐dependent inactivation of the C25S/C50S/C455S/C537S mutant by a number of squalene analogues and other agents with thiol‐modifying activity suggested that modifying C435 caused the obstruction of the channel constriction thus blocking access of the substrate to the active site. The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC‐ESI‐MS measurements. The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate‐protonating residue, was substituted by cysteine. The inhibitory effect was evaluated in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene. Among the inhibitors tested, the substrate analogue squalene‐maleimide proved to be a very effective time‐dependent inhibitor.
A pool of Y2 neuropeptide Y receptors activated by modifiers of membrane sulfhydryl or cholesterol balance
Authors: Parker Steven L. , Parker Michael S. , Kane Justin K. , Berglund Magnus M.
Published: March 2003

The cloned guinea‐pig Y2 neuropeptide Y (NPY) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the Y2 receptors natively expressed in rat forebrain, are distributed in two populations. A smaller population that is readily accessed by agonist peptides on the surface of intact cells constitutes less than 30% of Y2 receptors detected in particulates after cell homogenization. A much larger fraction of cell surface Y2 sites can be activated by sulfhydryl modifiers. A fast and large activation of these masked or cryptic sites could be obtained with membrane‐permeating, vicinal cysteine‐bridging arsenical phenylarsine oxide. A lower activation is effected by N‐ethylmaleimide, an alkylator that slowly penetrates lipid bilayers. The restricted‐access alkylator, 2‐[(trimethylammonium)ethyl]methanethiosulfonate, was not effective in unmasking these sites. Some of the hidden cell surface Y2 sites could be activated by polyene filipin III through complexing of membrane cholesterol. The results are consistent with the presence of a large Y2 reserve in a compartment that can be accessed by alteration of sulfhydryl balance or fluidity of the cell membrane, and by␣treatments that affect the anchoring and aggregation of membrane proteins.
Cloning and expression of two novel aldo‐keto reductases from Digitalis purpurea leaves
Authors: Gavidia Isabel , Pérez‐Bermúdez Pedro , Seitz H Ulrich
Published: March 2003

The aldo‐keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon–carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf‐specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Δ4‐3‐ketosteroid 5β‐reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68–80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA‐blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat‐shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.
Purification and characterization of VanXYC, a d,d‐dipeptidase/d,d‐carboxypeptidase in vancomycin‐resistant Enterococcus gallinarum BM4174
Authors: Podmore Adrian H. B. , Reynolds Peter E.
Published: March 2003

VanXYC, a bifunctional enzyme from VanC‐phenotype Enterococcus gallinarum BM4174 that catalyses d,d‐peptidase and d,d‐carboxypeptidase activities, was purified as the native protein, as a maltose‐binding protein fusion and with an N‐terminal tag containing six histidine residues. The kinetic parameters of His6–VanXYC were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for d‐Ala‐d‐Ala, the Km was 3.6 mm and kcat, 2.5 s−1; for UDP‐MurNAc‐L‐Ala‐d‐Glu‐L‐Lys‐d‐Ala‐d‐Ala (UDP‐MurNAc‐pentapeptide[Ala]), Km was 18.8 mm and kcat 6.2 s−1; for d‐Ala‐d‐Ser, Km was 15.5 mm and kcat 0.35 s−1. His6–VanXYC was inactive against the peptidoglycan precursor UDP‐MurNAc‐l‐Ala‐d‐Glu‐L‐Lys‐d‐Ala‐d‐Ser (UDP‐MurNAc‐pentapeptide[Ser]). The rate of hydrolysis of the terminal d‐Ala of UDP‐MurNAc‐pentapeptide[Ala] was inhibited 30% by 2 mm d‐Ala‐d‐Ser or UDP‐MurNAc‐pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in d‐Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in d‐Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.

Mutation of an aspartate residue (Asp59) of His‐tagged VanXYC corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (kcat/Km) for d‐Ala‐d‐Ala. This situation is in contrast to VanX in which mutation of Asp68→Ala produced a greater than 200 000‐fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.
Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation
Authors: Wu Peng , Nakano Shu‐ichi , Sugimoto Naoki
Published: March 2003

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non‐two‐state transition and heat capacity change. Although the temperature‐dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12 bp, were selected to evaluate the heat capacity changes and temperature‐dependent thermodynamic properties of duplex formation. Direct comparison reveals that the temperature‐independent thermodynamic parameters could provide a reasonable approximation only when the temperature of interest has a small deviation from the mean melting temperature over the experimental range. The heat capacity changes depend on the base composition and sequences and are generally limited in the range of −160 to ≈−40 cal·mol−1·K−1 per base pair. In contrast to the enthalpy and entropy changes, the free energy change and melting temperature are relatively insensitive to the heat capacity change. Finally, the 16 NN‐model free energy parameters and one helix initiation at physiological temperature were extracted from the temperature‐dependent thermodynamic data of the 41 RNA/DNA hybrids.
Phosphorylation of initiation factor‐2α is required for activation of internal translation initiation during cell differentiation
Authors: Gerlitz Gabi , Jagus Rosemary , Elroy‐Stein Orna
Published: March 2003

The long uORF‐burdened 5′UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet‐derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES‐mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c‐Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES‐mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2α. Furthermore, IRES use is decreased by over‐expression of the dominant‐negative form of the eIF2α kinase, PKR, the vaccinia virus K3L gene, or the eIF2α‐S51A variant which result in decreased eIF2α phosphorylation. These data demonstrate a connection between eIF2α phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2α, known to be important for cap‐dependent transaltional control, serves to fine‐tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES‐containing mRNAs under a range of physiological circumstances.
Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4
Authors: Desseyn Jean‐Luc , Clavereau Isabelle , Laine Anne
Published: March 2003

We report here the full coding sequence of a novel mouse putative membrane‐associated mucin containing three extracellular EGF‐like motifs and a mucin‐like domain consisting of at least 20 tandem repeats of 124–126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of ≈ 52 kb. The first intron is ≈ 16 kb in length and is followed by an unusually large exon (≈ 9.5 kb) encoding Ser/Thr‐rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane‐anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract.
A Ca2+/CaM‐dependent kinase from pea is stress regulated andin vitrophosphorylates a protein that binds toAtCaM5promoter
Authors: Pandey Sona , Tiwari Shiv B. , Tyagi Wricha , Reddy Mali K. , Upadhyaya Kailash C. , Sopory Sudhir K.
Published: March 2003

An immuno‐homologue of maize Ca2+/calmodulin (CaM)‐dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+application increased the kinase level and the response was faster than that obtained following stress application. Low temperature‐mediated, but not salinity‐mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno‐affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40in vitro. Gel mobility shift and South‐Western analysis showed that p40 is a DNA‐binding protein and it interacted specifically with one of thecisacting elements of theArabidopsisCaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in theAtCaM5promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.
Towards the development of selective amine oxidase inhibitors
Authors: Shepard Eric M. , Smith Jennifer , Elmore Bradley O. , Kuchar Jason A. , Sayre Lawrence M. , Dooley David M.
Published: March 2003

Four substrate analogs, 4‐(2‐naphthyloxy)‐2‐butyn‐1‐amine (1), 1,4‐diamino‐2‐chloro‐2‐butene (2), 1,6‐diamino‐2,4‐hexadiyne (3), and 2‐chloro‐5‐phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper‐containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4‐diamino‐2‐butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism‐based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side‐chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.
Phosphorylation of Hrs downstream of the epidermal growth factor receptor
Authors: Bache Kristi G. , Raiborg Camilla , Mehlum Anja , Madshus Inger Helene , Stenmark Harald
Published: March 2003

The hepatocyte growth factor‐regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src‐, Yes‐ and Fyn‐negative cells. Moreover, we show that only 10–20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.
Inhibition of SERCA Ca2+ pumps by 2‐aminoethoxydiphenyl borate (2‐APB)
Authors: Bilmen Jonathan G. , Wootton Laura L. , Godfrey Rita E. , Smart Oliver S. , Michelangeli Francesco
Published: March 2003

2‐Aminoethoxydiphenyl Borate (2‐APB) has been extensively used recently as a membrane permeable modulator of inositol‐1,4,5‐trisphosphate‐sensitive Ca2+ channels and store‐operated Ca2+ entry. Here, we report that 2‐APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2‐APB onthe SERCA Ca2+ pumps is isoform‐dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 µm, respectively, measured at pH 7.2). The Ca2+‐ATPase is also more potently inhibited at lower pH (IC50 = 70 µm for SERCA1A at pH 6). 2‐APB decreases the affinity for Ca2+ binding to the ATPase by more than 20‐fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+‐ATPases show that Tyr837 is critical for the inhibition of activity by 2‐APB. Molecular modeling studies of 2‐APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2‐APB to these sites could influence the movement of the loop between M6 and M7 (L6‐7), and reduce access of Ca2+ to their binding sites.
Structural study on lipid A and the O‐specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease
Authors: Hashimoto Masahito , Kirikae Fumiko , Dohi Taeko , Adachi Seizi , Kusumoto Shoichi , Suda Yasuo , Fujita Tsuyoshi , Naoki Hideo , Kirikae Teruo
Published: March 2003

Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O‐antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel‐filtration chromatography to give an OPS‐rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [→4)α‐l‐Rhap(1→3)β‐d‐Manp(1→].
Control of p70 ribosomal protein S6 kinase and acetyl‐CoA carboxylase by AMP‐activated protein kinase and protein phosphatases in isolated hepatocytes
Authors: Krause Ulrike , Bertrand Luc , Hue Louis
Published: March 2003

Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl‐CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP‐activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino‐acid‐induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5‐aminoimidazole‐4‐carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase‐dependent mechanism but did not modify AMPK activation. Conversely, the amino‐acid‐induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino‐acid‐induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy‐consuming biosynthetic pathways.
Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors
Authors: Avila Sonia , Casero Marie‐Carmen , Fernandez‐Cantón Rocío , Sastre Leandro
Published: March 2003

The transcription factor serum response factor (SRF) regulates expression of growth factor‐dependent genes and muscle‐specific genes in vertebrates. Homologous factors regulate differentiation of some ectodermic tissues in invertebrates. To explore the molecular basis of these different physiological functions, the functionality of human, Drosophila melanogaster and Artemia franciscana SRFs in mammalian cells has been compared in this article. D. melanogaster and, to a lesser extend, A. franciscana SRF co‐expression represses the activity of strong SRF‐dependent promoters, such as those of the mouse c‐fos and A. franciscana actin 403 genes. Domain‐exchange experiments showed that these results can be explained by the absence of a transactivation domain, functional in mammalian cells, in D. melanogaster and A. franciscana SRFs. Both invertebrate SRFs can dimerize with endogenous mouse SRF through the conserved DNA‐binding and dimerization domain. Co‐expression of human and A. franciscana SRFs activate expression of weaker SRF‐dependent promoters, such as those of the human cardiac α‐actin gene or an A. franciscana actin 403 promoter where the SRF‐binding site has been mutated. Mapping of A. franciscana SRF domains involved in transcriptional activation has shown that the conserved DNA‐binding and dimerization domain is neccessary, but not sufficient, for promoter activation in mammalian cells.
The proximal cis‐acting elements Sp1, Sp3 and E2F regulate mouse mer gene transcription in Sertoli cells
Authors: Wong Connie C. S. , Lee Will M.
Published: March 2003

Mer belongs to the Tyro 3 family of receptor tyrosine kinases (RTKs). Together with Axl and Rse, the three RTKs are believed to play important functional roles in the male gonads because gene knockout male mice lacking all of these receptors are infertile. In the present study, postnatal expression of Axl and Rse in mouse testes decreased during maturation while expression of Mer increased age‐dependently during testicular development. To investigate the transcriptional regulation of gene expression in the testis, a ≈ 1.5 kb fragment of the 5′ flanking sequence of Mer was isolated. The sequence lacks a typical TATA or CAAT box. 5′ RACE revealed that the putative major transcriptional start site of Mer is located at +102 bp upstream of the translation initiation site. Using transient transfections of luciferase reporter constructs driven by various lengths of the 5′ flanking sequence, the gene segment −321/+126 showed the highest transcriptional activity in a mouse Sertoli cell line (TM4). DNAase I footprinting experiments revealed four footprints within the region from −321 to −26, including three binding sites for the transcriptional factor Specificity protein 1 (Sp1) and one for an unknown transcriptional factor. Electrophoretic mobility shift assay (EMSA), supershift assay, mutation studies and cotransfection demonstrated that those Sp1 cis‐acting motifs interacted either with Sp1 or Sp1/Sp3, depending on location and the nearby nucleotide sequences. An E2F binding site which down‐regulates Mer transcription, as revealed by EMSA, deletion and mutation studies, was identified downstream in the proximity of the promoter. Taking all of these data together, the study has demonstrated that Sp1, Sp3, E2F and probably another unknown transcriptional factor play a critical role in regulating the proximal promoter activities of Mer.
Cloning of a rat gene encoding the histo‐blood group A enzyme
Authors: Cailleau‐Thomas Anne , Le Moullac‐Vaidye Béatrice , Rocher Jézabel , Bouhours Danièle , Szpirer Claude , Le Pendu Jacques
Published: March 2003

The complete coding sequence of a BDIX rat gene homologous to the human ABO gene was determined. Identification of the exon–intron boundaries, obtained by comparison of the coding sequence with rat genomic sequences from data banks, revealed that the rat gene structure is identical to that of the human ABO gene. It localizes to rat chromosome 3 (q11‐q12), a region homologous to human 9q34. Phylogenetic analysis of a set of sequences available for the various members of the same gene family confirmed that the rat sequence belongs to the ABO gene cluster. The cDNA was transfected in CHO cells already stably transfected with an α1,2fucosyltransferase in order to express H oligosaccharide acceptors. Analysis of the transfectants by flow cytometry indicated that A but not B epitopes were synthesized. Direct assay of the enzyme activity using 2′ fucosyllactose as acceptor confirmed the strong UDP‐GalNAc:Fucα1,2GalαGalNAc transferase (Atransferase) activity of the enzyme product and allowed detection of a small UDP‐Gal:Fucα1,2GalαGal transferase (B transferase) activity. The presence of the mRNA and of the A and B antigens was searched in various BDIX rat tissues. There was a general good concordance between the presence of the mRNA and that of the A antigen. Tissue distributions of the A and B antigens in the homozygous BDIX rat strain were largely different, indicating that these antigens cannot be synthesized by alleles of the same gene in this rat inbred strain.
Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials
Authors: Saloheimo Markku , Paloheimo Marja , Hakola Satu , Pere Jaakko , Swanson Barbara , Nyyssönen Eini , Bhatia Amit , Ward Michael , Penttilä Merja
Published: March 2003

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N‐terminal fungal type cellulose binding domain connected by a linker region to the expansin‐like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non‐stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin‐like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI‐containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.
Characterization of the 5′ untranslated region of α and β isoforms of the human thromboxane A2 receptor (TP)
Authors: Coyle Adrian T. , Miggin Sinead M. , Kinsella B. Therese
Published: March 2003

In humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, TPα and TPβ, that diverge within their carboxyl terminal cytoplasmic (C) tail regions and arise by differential splicing. The human TP gene contains three exons E1–E3; while E1 exclusively encodes 5′ untranslated region (UTR) sequence, E2 and E3 represent the main coding exons. An additional noncoding exon, E1b was identified within intron 1. Additionally, the TP gene contains two promoters P1 and P2 located 5′ of E1 and E1b, respectively.

Herein, we investigated the molecular basis of the differential expression of the TP isoforms by characterizing the 5′ UTR of the TP transcripts. While E1 and E1b were found associated with TP transcript(s), their expression was mutually exclusive. 5′ rapid amplification of cDNA ends (5′ RACE) established that the major transcription initiation (TI) sites were clustered between −115 and −92 within E1 and at −99 within E1b. While E1 and E1b sequences were identified on TPα transcript(s), neither existed on TPβ transcript(s). More specifically, TPα and TPβ transcripts diverged within E2 and the major TI sites for TPβ transcripts mapped to −12/−15 therein. Through genetic reporter assays, a previously unrecognized promoter, termed P3, was identified on the TP gene located immediately 5′ of −12. The proximity of P3 to the TI site of TPβ suggests a role for P3 in the control of TPβ expression and implies that TPα and TPβ, in addition to being products of differential splicing, are under the transcriptional control of distinct promoters.
A novel tachykinin‐related peptide receptor
Authors: Kawada Tsuyoshi , Furukawa Yasuo , Shimizu Yoriko , Minakata Hiroyuki , Nomoto Kyosuke , Satake Honoo
Published: March 2003

Structurally tachykinin‐related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G‐protein‐coupled receptor (GPCR) for a tachykinin‐related peptide. In this paper, we report the identification of a novel tachykinin‐related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin‐related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin‐related peptide receptors, activates calcium‐dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru‐TKs) I–V and VII, which were isolated as Urechis tachykinin‐related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru‐TKs equivalently, showing that UTKR possesses no selective affinity with Uru‐TKs. In contrast, UTKR was not activated by substance P or an Uru‐TK analog containing a C‐terminal Met‐NH2 instead of Arg‐NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron‐inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin‐related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin‐related peptide receptor from other species than insects and also of the genomic structure of a tachykinin‐related peptide receptor gene.
Electrostatic role of aromatic ring stacking in the pH‐sensitive modulation of a chymotrypsin‐type serine protease, Achromobacter protease I
Authors: Shiraki Kentaro , Norioka Shigemi , Li Shaoliang , Yokota Kiyonobu , Sakiyama Fumio
Published: March 2003

Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169–His210 in close proximity to the catalytic triad. This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5–10) among chymotrypsin‐type serine proteases. The pH‐activity profile of API showed a sigmoidal distribution that appears at pH 8–10, with a shoulder at pH 6–8. Variants with smaller amino acid residues substituted for Trp169 had lower pH optima on the acidic side by 0–0.9 units. On the other hand, replacement of His210 by Ala or Ser lowered the acidic rim by 1.9 pH units, which is essentially identical to that of chymotrypsin and trypsin. Energy minimization for the mutant structures suggested that the side‐chain of Trp169 stacked with His210 was responsible for isolation of the electrostatic interaction between His210 and the catalytic Asp113 from solvent. The aromatic stacking regulates the low activity at neutral pH and the high activity at alkaline pH due to the interference of the hydrogen bonded network in the catalytic triad residues.
Amyloidogenic nature of spider silk
Authors: Kenney John M. , Knight David , Wise Michael J. , Vollrath Fritz
Published: March 2003

In spiders soluble proteins are converted to form insoluble silk fibres, stronger than steel. The final fibre product has long been the subject of study; however, little is known about the conversion process in the silk‐producing gland of the spider. Here we describe a study of the conversion of the soluble form of the major spider‐silk protein, spidroin, directly extracted from the silk gland, to a β‐sheet enriched state using circular dichroism (CD) spectroscopy. Combined with electron microscopy (EM) data showing fibril formation in the β‐sheet rich region of the gland and amino‐acid sequence analyses linking spidroin and amyloids, these results lead us to suggest that the refolding conversion is amyloid like. We also propose that spider silk could be a valuable model system for testing hypotheses concerning β‐sheet formation in other fibrilogenic systems, including amyloids.
Short‐chain dehydrogenases/reductases (SDRs)
Authors: Kallberg Yvonne , Oppermann Udo , Jörnvall Hans , Persson Bengt
Published: March 2003

Short‐chain dehydrogenases/reductases (SDRs) are enzymes of great functional diversity. Even at sequence identities of typically only 15–30%, specific sequence motifs are detectable, reflecting common folding patterns. We have developed a functional assignment scheme based on these motifs and we find five families. Two of these families were known previously and are called ‘classical’ and ‘extended’ families, but they are now distinguished at a further level based on coenzyme specificities. This analysis gives seven subfamilies of classical SDRs and three subfamilies of extended SDRs. We find that NADP(H) is the preferred coenzyme among most classical SDRs, while NAD(H) is that preferred among most extended SDRs. Three families are novel entities, denoted ‘intermediate’, ‘divergent’ and ‘complex’, encompassing short‐chain alcohol dehydrogenases, enoyl reductases and multifunctional enzymes, respectively. The assignment scheme was applied to the genomes of human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae. In the animal genomes, the extended SDRs amount to around one quarter or less of the total number of SDRs, while in the A. thaliana and S. cerevisiae genomes, the extended members constitute about 40% of the SDR forms. The numbers of NAD(H)‐dependent and NADP(H)‐dependent SDRs aresimilar in human, mouse and plant, while the proportions of NAD(H)‐dependent enzymes are much lower in fruit fly, worm and yeast. We show that, in spite of the great diversity of the SDR superfamily, the primary structure alone can be used for functional assignments and for predictions of coenzyme preference.
Sendai virus N‐terminal fusion peptide consists of two similar repeats, both of which contribute to membrane fusion
Authors: Peisajovich Sergio G. , Epand Raquel F. , Epand Richard M. , Shai Yechiel
Published: March 2003

The N‐terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N‐terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N‐terminus, this segment (WT‐palm‐19–33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N‐terminal fusion peptide. The activity of WT‐palm‐19–33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino‐acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1‐ induced cell‐cell fusion, also increased the homology between the N‐terminal fusion peptide and WT‐palm‐19–33. The role of the amino‐acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1–33), a G12A mutant (G12A 1–33), a G7A–G12A double mutant (G7A–G12A 1–33), and a peptide with a scrambled sequence (SC 1–33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the α helical content of the N‐terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33‐residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.
NMR and MS evidences for a random assembled O‐specific chain structure in the LPS of the bacterium Xanthomonas campestris pv. Vitians
Authors: Molinaro Antonio , De Castro Cristina , Lanzetta Rosa , Parrilli Michelangelo , Petersen Bent O , Broberg Anders , Duus Jens Ø
Published: March 2003

Xanthomonas campestris pv. vitians is a Gram‐negative plant‐associated bacterium that acts as causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide of this bacterium is suspected to be an important molecule for adhesion to and infection of the plants. The lipopolysaccharide has been isolated from the phenol phase and the O‐specific chain characterized by compositional analysis, high field NMR and MALDI‐TOF MS. It consists of a nonrepetitive branched polysaccharide with a rhamnan backbone to which Fuc3NAc is linked. The NMR and MS approach led to the characterization of the fine structure of the polymer, which is randomly assembled. The rhamnan backbone is built up of β‐Rhap and α‐Rhap, this last is present in one, two or three adjacent units and branched by an α‐Fucp3NAc unit. This is a real case of a random constituted O‐specific chain, therefore biosynthetic studies towards the comprehension of this irregular biosynthesis are needed.
Modeling the three‐dimensional structure of H+‐ATPase of Neurospora crassa
Authors: Radresa Olivier , Ogata Koji , Wodak Shoshana , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+‐ATPase, using as template the 2.6 Å crystal structure of rabbit sarcoplasmic reticulum Ca2+‐ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647–655]. Comparison of the two calcium‐binding sites in the crystal structure of Ca2+‐ATPase with the equivalent region in the H+‐ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high‐resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+‐ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.
Mammalian mitochondrial endonuclease G
Authors: Ohsato Takashi , Ishihara Naotada , Muta Tsuyoshi , Umeda Shuyo , Ikeda Shogo , Mihara Katsuyoshi , Hamasaki Naotaka , Kang Dongchon
Published: March 2003

Mammalian mitochondria contain strong nuclease activity. Endonuclease G (endoG), which predominantly resides in mitochondria, accounts for a large part of this nuclease activity. It has been proposed to act as an RNase H‐like nuclease on RNA·DNA hybrids (R‐loops) in the D‐loop region where the origins of mitochondrial replication are mapped, providing RNA primers for mtDNA replication. However, in contrast with this proposed activity, endoG has recently been shown to translocate to nuclei on apoptotic stimulation and act as a nuclease without sequence specificity. To clarify the role of endoG in mtDNA replication, we examined its submitochondrial localization and its ability to cleave R‐loops. At low concentration, it preferentially produces double‐stranded breaks in R‐loops, but does not act as an RNase H‐like nuclease. In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs. These results do not support the involvement of endoG in mtDNA replication. Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double‐stranded breaks in R‐loops, we propose that three‐stranded DNA may be the preferred substrate of endoG.
A polymer with a backbone of 3‐deoxy‐d‐glycero‐d‐galacto‐non‐2‐ulopyranosonic acid, a teichuronic acid, and a β‐glucosylated ribitol teichoic acid in the cell wall of plant pathogenic Streptomyces sp. VKM Ac‐2124
Authors: Shashkov Alexander S. , Kosmachevskaya Larisa N. , Streshinskaya Galina M. , Evtushenko Lyudmila I. , Bueva Olga V. , Denisenko Viktor A. , Naumova Irina B. , Stackebrandt Erko
Published: March 2003

Structures of cell wall anionic polymers of the strain Streptomyces sp. VKM Ac‐2124, a causative agent of potato scab, which is phylogenetically the closest to plant pathogenic species S. setonii and S. caviscabies, were studied. The strain contains three anionic glycopolymers, viz., a teichuronic acid with a disaccharide repeating unit →6)‐α‐d‐Glcp‐(1→4)‐β‐d‐ManpNAc3NAcA‐(1→, a β‐glucosylated polymer of 3‐deoxy‐d‐glycero‐d‐galacto‐non‐2‐ulopyranosonic acid (Kdn), and a β‐glucosylated 1,5‐poly(ribitol phosphate). The strain studied is the second representative of plant pathogenic streptomycetes inducing potato scab disease, the cell wall anionic polymers of which were shown to contain a Kdn‐polymer. Presumably, the presence of Kdn‐containing structures in the surface regions of pathogens is essential for their efficient attachment to host plant cells.