bluuebunny/crossref_metadata_2025 huggingface dataset:
O‐acetylation and de‐O‐acetylation of sialic acids in human colorectal carcinoma
Authors: Shen Yanqin , Kohla Guido , Lrhorfi Aicha L. , Sipos Bence , Kalthoff Holger , Gerwig Gerrit J. , Kamerling Johannis P. , Schauer Roland , Tiralongo Joe
Published: January 2004

A decrease in the level of O‐acetylated sialic acids observed in colorectal carcinoma may lead to an increase in the expression of sialyl LewisX, a tumor‐associated antigen, which is related to progression of colorectal cancer to metastasis. The underlying mechanism for this reduction is, however, not fully understood. Two enzymes are thought to be primarily responsible for the turnover of O‐acetyl ester groups on sialic acids; sialate‐O‐acetyltransferase (OAT) and sialate‐O‐acetylesterase (OAE). We have previously reported the characterization of OAT activity from normal colon mucosa, which efficiently O‐acetylates CMP‐Neu5Ac exclusively in the Golgi apparatus prior to the action of sialyltransferase [Shen, Y., Tiralongo, J., Iwersen, M., Sipos, B., Kalthoff, H. & Schauer, R. (2002) Biol. Chem.383, 307–317]. In this report we describe the identification of a lysosomal and a cytosolic OAE activity in human colonic mucosa that specifically hydrolyses 9‐O‐acetyl groups on sialic acid. Utilizing matched resection margin and cancer tissue from colorectal carcinoma patients we provide strong evidence suggesting that the level of O‐acetylated sialic acids present in normal and diseased human colon may be dependent on the relative activities of OAT to lysosomal OAE. Furthermore, we show that the level of free cytosolic Neu5,9Ac2 in human colon is regulated by the relative activity of the cytosolic OAE.
Some properties of human small heat shock protein Hsp20 (HspB6)
Authors: Bukach Olesya V. , Seit‐Nebi Alim S. , Marston Steven B. , Gusev Nikolai B.
Published: January 2004

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide‐dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55–60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45–50 and 28–30 kDa. At pH 7.0–7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction‐induced aggregation of insulin or heat‐induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial α‐crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild‐type protein. At pH 6.0, both α‐crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, α‐crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat‐induced aggregation of alcohol dehydrogenase. The mixing of wild‐type human Hsp27 and Hsp20 resulted in a slow, temperature‐dependent formation of hetero‐oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase‐activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero‐oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.
Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells
Authors: Legrand Dominique , Vigié Keveen , Said Elias A. , Elass Elisabeth , Masson Maryse , Slomianny Marie‐Christine , Carpentier Mathieu , Briand Jean‐Paul , Mazurier Joël , Hovanessian Ara G.
Published: January 2004

Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf‐binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf‐binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium–affinity interactions (≈ 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface‐expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti‐human immunodeficiency virus pseudopeptide, HB‐19, which binds specifically surface‐expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB‐19 and various Lf derivatives in proteoglycan‐deficient hamster cells suggested that the nucleolin‐binding site is located in both the N‐ and C‐terminal lobes of Lf, whereas the basic N‐terminal region is dispensable. On intact cells, Lf co‐localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB‐19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan‐deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.
Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea
Authors: Chen Shicheng , Ge Wei , Buswell John A.
Published: January 2004

We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion‐exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N‐terminal sequence of purified lac1 and a conserved copper‐binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full‐length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N‐terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT‐PCR analysis of gene transcription in fungal mycelia grown on rice‐straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ≈ 20–30% of peak levels throughout the subsequent stages of sporophore development.
A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii
Authors: Pineau Bernard , Girard‐Bascou Jacqueline , Eberhard Stephan , Choquet Yves , Trémolières Antoine , Gérard‐Hirne Catherine , Bennardo‐Connan Annick , Decottignies Paulette , Gillet Sylvie , Wollman Francis‐André
Published: January 2004

Two mutants of Chlamydomonas reinhardtii, mf1 and mf2, characterized by a marked reduction in their phosphatidylglycerol content together with a complete loss in its Δ3‐trans hexadecenoic acid‐containing form, also lost photosystem II (PSII) activity. Genetic analysis of crosses between mf2 and wild‐type strains shows a strict cosegregation of the PSII and lipid deficiencies, while phenotypic analysis of phototrophic revertant strains suggests that one single nuclear mutation is responsible for the pleiotropic phenotype of the mutants. The nearly complete absence of PSII core is due to a severely decreased synthesis of two subunits, D1 and apoCP47, which is not due to a decrease in translation initiation. Trace amounts of PSII cores that were detected in the mutants did not associate with the light‐harvesting chlorophyll a/b‐binding protein antenna (LHCII). We discuss the possible role of phosphatidylglycerol in the coupled process of cotranslational insertion and assembly of PSII core subunits.
Spatio‐temporal distribution of cellular retinol‐binding protein gene transcripts (CRBPI and CRBPII) in the developing and adult zebrafish (Danio rerio)
Authors: Liu Rong‐Zong , Denovan‐Wright Eileen M. , Degrave Agnes , Thisse Christine , Thisse Bernard , Wright Jonathan M.
Published: January 2004

We have cloned and determined the nucleotide sequence of the cDNA coding for a cellular retinol‐binding protein type I (CRBPI) from zebrafish. The deduced amino acid sequence of the zebrafish CRBPI showed highest sequence identity (≈ 59%) to the mammalian CRBPIs of the intracellular lipid‐binding protein (iLBP) multigene family. Phylogenetic analysis clustered the zebrafish CRBPI to the CRBPI clade. The zebrafish CRBPI gene (rbp1) and CRBPII gene (rbp2) both consist of four exons separated by three introns, identical to all other iLBP genes in vertebrates. Two transcription start sites were identified in the rbp1 promoter and a single transcription start site was identified for rbp2. Radiation hybrid mapping assigned the zebrafish rbp1 gene to linkage group 16 and conserved syntenic genes were found by comparative analysis of mammalian orthologous rbp1 genes. RT‐PCR detected mRNA transcripts in the adult intestine, liver, brain, ovary and testis for rbp1 gene and in the intestine and liver for rbp2 gene. Whole mount in situ hybridization of zebrafish embryos revealed rbp1 mRNA expression in the developing zebrafish central nervous system at specific sites that are known to have abundant retinoic acid distribution and significant retinoic acid action. Whole mount in situ hybridization also showed that the zebrafish rbp2 mRNA was localized specifically in the embryonic intestinal bulb and the developing intestine during the larval stage, implying a novel function for the rbp2 gene product during organogenesis and development of the zebrafish intestine.
GTP cyclohydrolase I utilizes metal‐free GTP as its substrate
Authors: Suzuki Takahiro , Kurita Hideki , Ichinose Hiroshi
Published: January 2004

GTP cyclohydrolase I (GCH) is the rate‐limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg‐free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal‐free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg‐free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.
Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L‐chain for protection of α1,2‐mannose residues in N‐linked oligosaccharide chains of fibrohexamerin/P25
Authors: Inoue Satoshi , Tanaka Kazunori , Tanaka Hiromitsu , Ohtomo Kohei , Kanda Toshio , Imamura Morikazu , Quan Guo‐Xing , Kojima Katsura , Yamashita Tetsuro , Nakajima Tasuku , Taira Hideharu , Tamura Toshiki , Mizuno Shigeki
Published: January 2004

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3‐MDa elementary unit, consisting of six sets of a disulfide‐linked heavy chain (H‐chain)–light chain (L‐chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H–L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal‐level fibroin‐producing breeds (J‐139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27‐kDa fhx/P25 was produced from the 30‐kDa form by digestion with the bacterial α1,2‐mannosidase in vitro. The elementary unit in the ER extract contained only the 30‐kDa fhx/P25, whereas both 30‐ and 27‐kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked‐pupa mutants [Nd(2), Nd‐s and Nd‐sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27‐kDa fhx/P25 and six molecules of H‐chain but no L‐chain. When the Nd‐sD mutant was subjected to transgenesis with the normal L‐chain gene, the (H‐L)6fhx1‐type elementary unit containing the 30‐kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi α1,2‐mannosidases when L‐chains are present in the unit. Models suggesting a role of L‐chain for the protection of α1,2‐mannose residues of fhx/P25 are presented.
Mycoplasma pneumoniae HPr kinase/phosphorylase
Authors: Merzbacher Matthias , Detsch Christian , Hillen Wolfgang , Stülke Jörg
Published: January 2004

HPr kinase/phosphorylase (HPrK/P) is the key regulator of carbon metabolism in many Gram‐positive bacteria. It phosphorylates/dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae, HPrK/P is one of the very few regulatory proteins encoded in the genome. The regulation of this enzyme by metabolites is unique among HPrK/P proteins studied so far: it is active as a kinase at low ATP concentrations, whereas the proteins from other bacteria need high ATP concentrations as an indicator of a good nutrient supply for kinase activity. We studied the interaction of M. pneumoniae HPrK/P with ATP, Fru1,6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation, we found a very high affinity for ATP (Kd = 5.4 µm) compared with the HPrK/P proteins from other bacteria. The Kd for Fru1,6P2 was three orders of magnitude higher, which explains why Fru1,6P2 has only a weak regulatory effect on M. pneumoniae HPrK/P. Mutations of two important regions in the active site of HPrK/P, the nucleotide binding P‐loop and the HPrK/P family signature sequence, had different effects. P‐loop region mutations strongly affect ATP binding and thus all enzymatic functions, whereas the signature sequence motif seems to be important for the catalytic mechanism rather than for nucleotide binding.
Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations
Authors: Culvenor Janetta G. , Ilaya Nancy T. , Ryan Michael T. , Canterford Louise , Hoke David E. , Williamson Nicholas A. , McLean Catriona A. , Masters Colin L. , Evin Geneviève
Published: January 2004

The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N‐ and C‐terminal fragments in association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1. Solubilization of mouse or human brain membranes with dodecyl‐d‐maltoside produced a 360‐kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X‐100 and SDS, but stable to reducing agent. Addition of 5 m urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer‐2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second‐dimension analysis. Anterior pharynx defective‐1 was detected as an 18‐kDa form and 9‐kDa C‐terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36‐kDa band after presenilin 1 complex second‐dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1‐reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.
Binding analyses between Human PPARγ–LBD and ligands
Authors: Yu Changying , Chen Lili , Luo Haibing , Chen Jing , Cheng Feng , Gui Chunshan , Zhang Ruihao , Shen Jianhua , Chen Kaixian , Jiang Hualiang , Shen Xu
Published: January 2004

The binding characteristics of a series of PPARγ ligands (GW9662, GI 262570, cis‐parinaric acid, 15‐deoxy‐Δ12,14‐prostaglandin J2, LY171883, indomethacin, linoleic acid, palmitic acid and troglitazone) to human PPARγ ligand binding domain have been investigated for the first time by using surface plasmon resonance biosensor technology, CD spectroscopy and molecular docking simulation. The surface plasmon resonance biosensor determined equilibrium dissociation constants (KD values) are in agreement with the results reported in the literature measured by other methods, indicating that the surface plasmon resonance biosensor can assume a direct assay method in screening new PPARγ agonists or antagonists. Conformational changes of PPARγ caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (Tm), was enhanced by the binding of the ligands. The increment of the transition temperature (ΔTm) of PPARγ owing to ligand binding correlated well with the binding affinity. This finding implies that CD could possibly be a complementary technology with which to determine the binding affinities of ligands to PPARγ. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARγ at the atomic level, which gave a good explanation of the structure‐binding affinity relationship for the ligands interacting with PPARγ. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARγ ligands, although the docking program cannot accurately predict the absolute ligand‐PPARγ binding affinity.
Endoplasmic reticulum‐associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8‐5 CHO cell line
Authors: Foulquier François , Duvet Sandrine , Klein André , Mir Anne‐Marie , Chirat Frédéric , Cacan René
Published: January 2004

Endoplasmic reticulum‐associated degradation of newly synthesized glycoproteins has been demonstrated previously using various mammalian cell lines. Depending on the cell type, glycoproteins bearing Man9 glycans and glycoproteins bearing Man5 glycans can be efficiently degraded. A wide variety of variables can lead to defective synthesis of lipid‐linked oligosaccharides and, therefore, in mammalian cells, species derived from Man9GlcNAc2 or Man5GlcNAc2 are often recovered on newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line lacking Glc‐P‐Dol‐dependent glucosyltransferase I to generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins. By studying the structure of the soluble oligomannosides produced by degradation of these glycoproteins, we demonstrated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially.
Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody
Authors: Chark Davin , Nutikka Anita , Trusevych Natasha , Kuzmina Julia , Lingwood Clifford
Published: January 2004

The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin‐induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti‐globotriaosylceramide are distinct. Anti‐globotriaosylceramide had greater dependency on the intact α‐galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin‐associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti‐globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti‐globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl‐β‐cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate‐verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti‐fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate‐verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate‐verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti‐globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol‐dependent?) role in determining renal pathology following verotoxemia.
New substrate analogues of human serotonin N‐acetyltransferase produce in situ specific and potent inhibitors
Authors: Ferry Gilles , Ubeaud Caroline , Mozo Julien , Péan Christophe , Hennig Philippe , Rodriguez Marianne , Scoul Catherine , Bonnaud Anne , Nosjean Olivier , Galizzi Jean‐Pierre , Delagrange Philippe , Renard Pierre , Volland Jean‐Paul , Yous Said , Lesieur Daniel , Boutin Jean A.
Published: January 2004

Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N‐acetyltransferase catalyzes the rate‐limiting step. A previous study [Khalil, E.M., De Angelis, J., Ishii, M. & Cole, P.A. (1999) Proc. Natl Acad. Sci. USA96, 12418–12423] reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme. This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites. This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A. In the present report, we describe the potency of new N‐halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N‐acetyltransferase. The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated‐BAT in a living cell. The fate of tritiated‐phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of [3H]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N‐acetyl[3H]PEA and coenzyme A‐S[3H]acetyltryptamine, respectively). The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme. In a similar manner we studied the production of another inhibitor generated from N‐[2‐(7‐hydroxynaphth‐1‐yl)ethyl]bromoacetamide. New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model. Some of these compounds proved to be extremely potent, with IC50s of ≈ 30 nm. As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N‐acetyltransferase, we also show that they are potent ligands at the melatonin receptors. Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin production in vivo.
Mutational and structural analysis of cobalt‐containing nitrile hydratase on substrate and metal binding
Authors: Miyanaga Akimasa , Fushinobu Shinya , Ito Kiyoshi , Shoun Hirofumi , Wakagi Takayoshi
Published: January 2004

Mutants of a cobalt‐containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild‐type enzyme showed significantly lower Km and Ki values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n‐butyric acid) the hydroxyl group of βTyr68 formed hydrogen bonds with both n‐butyric acid and αSer112, which is located in the active center. The βY68F mutant showed an elevated Km value and a significantly decreased kcat value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between αCys108 and αCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt‐containing or iron‐containing nitrile hydratases. Two mutants (αT109S and αY114T) were constructed, each residue being replaced with an iron‐containing one. The αT109S mutant showed similar characteristics to the wild‐type enzyme. However, the αY114T mutant showed a very low cobalt content and catalytic activity compared with the wild‐type enzyme, and oxidative modifications of αCys111 and αCys113 residues were not observed. The αTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.
Crystal structure of the chi:psi subassembly of the Escherichia coli DNA polymerase clamp‐loader complex
Authors: Gulbis Jacqueline M. , Kazmirski Steven L. , Finkelstein Jeff , Kelman Zvi , O'Donnell Mike , Kuriyan John
Published: January 2004

The chi (χ) and psi (ψ) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP‐dependent clamp‐loader machinery. In E. coli, the χ:ψ heterodimer serves as a bridge between the clamp‐loader complex and the single‐stranded DNA‐binding protein. We determined the crystal structure of the χ:ψ heterodimer at 2.1 Å resolution. Although neither χ (147 residues) nor ψ (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide‐(χ) or dinucleotide‐(ψ) binding proteins, without marked similarity to the structures of the clamp‐loader subunits. Genes encoding χ and ψ proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the χ:ψ interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface‐exposed residues is restricted to the interfacial region and to just two other regions in the χ:ψ complex. One of the conserved regions was found to be located on χ, distal to the ψ interaction region, and we identified this as the binding site for a C‐terminal segment of the single‐stranded DNA‐binding protein. The other region of sequence conservation is localized to an N‐terminal segment of ψ (26 residues) that is disordered in the crystal structure. We speculate that ψ is linked to the clamp‐loader complex by this flexible, but conserved, N‐terminal segment, and that the χ:ψ unit is linked to the single‐stranded DNA‐binding protein via the distal surface of χ. The base of the clamp‐loader complex has an open C‐shaped structure, and the shape of the χ:ψ complex is suggestive of a loose docking within the crevice formed by the open faces of the δ and δ′ subunits of the clamp‐loader.
Human PABP binds AU‐rich RNA via RNA‐binding domains 3 and 4
Authors: Sladic Rosemary T. , Lagnado Cathy A. , Bagley Christopher J. , Goodall Gregory J.
Published: January 2004

Poly(A) binding protein (PABP) binds mRNA poly(A) tails and affects mRNA stability and translation. We show here that there is little free PABP in NIH3T3 cells, with the vast majority complexed with RNA. We found that PABP in NIH3T3 cytoplasmic lysates and recombinant human PABP can bind to AU‐rich RNA with high affinity. Human PABP bound an AU‐rich RNA with Kd in the nm range, which was only sixfold weaker than the affinity for oligo(A) RNA. Truncated PABP containing RNA recognition motif domains 3 and 4 retained binding to both AU‐rich and oligo(A) RNA, whereas a truncated PABP containing RNA recognition motif domains 1 and 2 was highly selective for oligo(A) RNA. The inducible PABP, iPABP, was found to be even less discriminating than PABP in RNA binding, with affinities for AU‐rich and oligo(A) RNAs differing by only twofold. These data suggest that iPABP and PABP may in some situations interact with other RNA regions in addition to the poly(A) tail.
Transitional Cell Carcinoma of the Renal Pelvis in Two Dogs
Authors: Militerno G. , Bazzo R. , Bevilacqua D. , Bettini G. , Marcato P. S.
Published: January 2004

Transitional cell carcinoma (TCC) of the renal pelvis was found in two dogs, a 7‐year‐old male English Setter and a 11‐year‐old female Shetland shepherd. Affected dogs were presented for clinical examination without any specific symptoms but haematuria in case 1 and occurrence of whitish material in the urine of case 2; neoplastic disorders were discovered with ultrasonographic investigation and fine needle aspiration biopsy. Histopathological examination was carried out after nephrectomy and ureterectomy of the affected kidney of both dogs, and confirmed the diagnosis of non‐invasive and low grade TCC in case 1 and of infiltrating TCC in case 2. The clinical, gross, cytological and histopathological features of these rare tumours originating from transitional epithelium of the renal pelvis are reported.
Sealing evaluation of the cervical base in intracoronal bleaching
Authors: De Oliveira Luciane Dias , Carvalho Claudio Antonio Talge , Hilgert Edson , Bondioli Ilda Ribeiro , De Araújo Maria Amélia Máximo , Valera Marcia Carneiro
Published: January 2004

Abstract –  Discoloration of non‐vital teeth is an esthetic deficiency frequently requiring bleaching treatment. The purpose of this study was to evaluate in vitro the cervical base efficacy in order to prevent or to minimize the leakage along the root canal filling and into the dentinal tubules. Thirty‐eight extracted single‐root human teeth were used, which were biomechanically prepared, filled, and divided into three experimental groups: G1, a cervical base was applied (3 mm of thickness) below the cemento‐enamel junction, with resin‐modified glass‐ionomer cement (Vitremer); G2, the base was done with glass‐ionomer cement (Vidrion R); and G3 (Control), did not receive any material as base. A mixture of sodium perborate and hydrogen peroxide 30% was placed inside the pulp chamber for 3 days, and the access opening was sealed with Cimpat. This procedure was repeated thrice. Soon after this, a paste of calcium hydroxide was inserted into the pulp chamber for 14 days. All teeth were covered with two layers of sticky wax, except the access opening, and immersed in blue India Ink for 5 days. The results did not show statistically significant differences between the three groups concerning the leakage inside the dentinal tubules. Regarding the apical direction, a statistical difference (anovaP < 0.05) was observed among the experimental group G1 and control group G3. No statistically significant difference was observed between G2 and G3 groups. Therefore, the placement of a cervical base before internal bleaching procedures is still recommended.
Traumatic dental injuries in Brazilian preschool children
Authors: Kramer Paulo Floriani , Zembruski Cíntia , Ferreira Simone Helena , Feldens Carlos Alberto
Published: January 2004

Abstract  – The purpose of this study was to determine the prevalence and distribution of traumatic injuries to primary anterior teeth in children from zero to 6 years old, all attendees in 28 public nursery schools in Canoas (Brazil). A total of 1545 boys and girls participated in the study. The children were clinically examined for signs of trauma according to Andreasen's classification. Traumatic injuries were identified in 35.5% of children. The largest percentage of injuries was demonstrated by 3–4‐year‐old child, with no significant difference between boys and girls. The maxillary central incisor was the most vulnerable to injury, without differences between the right and the left side. Single tooth injury was predominant in all age groups. Crown fractures represented 83% of all traumatic injuries. Our findings emphasize the importance of encouraging parents to visit the dentist with their child at an early stage. Furthermore, parents and educators should be advised about prevention of traumatic injuries and actions to be taken in case of an accident.
Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar
Authors: Salako Nathanael , Joseph Bobby , Ritwik Priyanshi , Salonen Jukka , John Preethi , Junaid T. A.
Published: January 2004

Abstract –  Bioactive glass (BAG) is often used as a filler material for repair of dental bone defects. Although there is evidence of osteogenic potential of this material, it is not clear yet whether the material exhibits potential for dentinogenesis. Hence, the aim of the present study was to evaluate BAG as a pulpotomy agent and to compare it with three commercially available pulpotomy agents such as formocresol (FC), ferric sulfate (FS), and mineral trioxide aggregate (MTA). Pulpotomies were performed in 80 maxillary first molars of Sprague Dawley rats, and pulp stumps were covered with BAG, FC, FS, and MTA. Histologic analysis was performed at 2 weeks and then at 4 weeks after treatment. Experimental samples were compared with contra‐lateral normal maxillary first molars. At 2 weeks, BAG showed inflammatory changes in the pulp. After 4 weeks, some samples showed normal pulp histology, with evidence of vasodilation. At 2 weeks, MTA samples showed some acute inflammatory cells around the material with evidence of macrophages in the radicular pulp. Dentine bridge formation with normal pulp histology was a consistent finding at 2 and 4 weeks with MTA. Ferric sulfate showed moderate inflammation of pulp with widespread necrosis in coronal pulp at 2 and 4 weeks. Formocresol showed zones of atrophy, inflammation, and fibrosis. Fibrosis was more extensive at 4 weeks with evidence of calcification in certain samples. Among the materials tested, MTA performed ideally as a pulpotomy agent causing dentine bridge formation while simultaneously maintaining normal pulpal histology. It appeared that BAG induced an inflammatory response at 2 weeks with resolution of inflammation at 4 weeks.
Etiological factors related to dental injuries in Norwegians aged 7–18 years
Authors: Skaare Anne B. , Jacobsen Ingeborg
Published: January 2004

Abstract  – Prevention of diseases and injuries is a field of priority for the health authorities in Norway. In 1990, an interdepartmental project was established in order to reduce the number of accidents, including dental injuries. The purpose of the present study was to examine the causes of dental injuries, the place of event and the possibilities for prevention. During a 1‐year period, all new trauma episodes affecting children aged 7–18 years were registered by calibrated dentists in the public dental health service in the county of Nord‐Trøndelag and in the capital city of Oslo. A distinction was made between school and leisure time accidents. The severity of injuries was classified as mild, moderate or severe, and the dentists were asked to judge whether the severe injuries were preventable. Nearly half of the 1275 injured individuals (48%) were injured at school. Almost half of the leisure time injuries occurred during children's play. Sports and traffic accidents were less common. Sports accidents represented only 8% of the total number of injuries, the same as the number of individuals injured by violence. Statistically significant associations were observed between the cause of injury and county (P < 0.001) and the cause of injury and age group (P < 0.001). In total, 4% of all injuries were classified as severe, of which only one‐third were assumed to be preventable. In summary, because of the complexity of etiological factors, it is neither easy to prevent dental injuries nor to make guidelines on prevention.
Intentional replantation with 180° rotation of a crown–root fracture: a case report
Authors: Fariniuk Luiz Fernando , Ferreira Erica Lopes , Soresini Gizelle C. Garcia , Cavali Ana Egide C. , Baratto Filho Flares
Published: January 2004

Abstract  – Crown–root fractures are frequently difficult to treat and have a bad prognosis. To restore the biological space, intentional replantation can be chosen when osteotomy/osteoplasty and dental traction are not viable. Intentional replantation with tooth rotation consists of extraction, 180° rotation and replantation, thus restoring the biological space. After treatment of inflammatory resorption with calcium hydroxide, endodontic treatment was completed. The patient was followed up with clinical examination (mobility test, gingival sulcus depth) and radiographic analysis (to analyze root resorption: inflammatory and/or substitution, and the integrity of the alveolar cortex and the periodontal space) for 3 years and there were no signs of resorption.
Electric field-assisted bipolar charge spouting in organic thin-film diodes
Authors: Tsutsui Tetsuo , Terai Masaya
Published: January 2004

We propose a concept of electric field-assisted bipolar charge spouting in organic solid films, and experimentally verify this concept based on device characteristics of organic thin-film diodes. Three organic thin-film diodes, indium-tin oxide (ITO)/N,N′-diphenyl-N,N′-bis(3-methylphenyl)-1,1′-biphenyl-4,4′-diamine (TPD)/tris(8-quinolinolato)alminum(III) (Alq3)/Al (conventional light-emitting diode), ITO/Alq3/TPD/Al (inverted-stack diode), and ITO/Alq3/Mg-doped Alq3/V2O5/TPD/Al (inverted-stack diode with carrier spouting zone), are prepared and their diode characteristics are observed. In the second diode, essentially no current flow is found when the ITO electrode is biased positive. On the other hand, large current flow is observed in the third diode, indicating the occurrence of charge carrier spouting from the charge separation zone composed of Mg-doped Alq3 and V2O5.
Demonstration of mammalian protein
O
-mannosyltransferase activity: Coexpression of POMT1 and POMT2 required for enzymatic activity
Authors: Manya Hiroshi , Chiba Atsuro , Yoshida Aruto , Wang Xiaohui , Chiba Yasunori , Jigami Yoshifumi , Margolis Richard U. , Endo Tamao
Published: January 2004

Defects in
O
-mannosylation of α-dystroglycan are thought to cause certain types of congenital muscular dystrophies with neuronal migration disorders. Among these muscular dystrophies, Walker–Warburg syndrome is caused by mutations in the gene encoding putative protein
O
-mannosyltransferase 1 (POMT1), which is homologous to yeast protein
O
-mannosyltransferases. However, there is no evidence that POMT1 has enzymatic activity. In this study, we first developed a method to detect protein
O
-mannosyltransferase activity in mammalian cells. Then, using this method, we showed that coexpression of both
POMT1
and
POMT2
(another gene homologous to yeast protein
O
-mannosyltransferases) was necessary for the enzyme activity, but expression of either
POMT1
or
POMT2
alone was insufficient. The requirement of an active enzyme complex of POMT1 and POMT2 suggests that the regulation of protein
O
-mannosylation is complex. Further, protein
O
-mannosylation appears to be required for normal structure and function of α-dystroglycan in muscle and brain. In view of the potential importance of this form of glycosylation for a number of developmental and neurobiological processes, the ability to assay mammalian protein
O
-mannosyltransferase activity should greatly facilitate progress in the identification and localization of
O
-mannosylated proteins and the elucidation of their functional roles.
The second extracellular loop of the dopamine D2receptor lines the binding-site crevice
Authors: Shi Lei , Javitch Jonathan A.
Published: January 2004

The binding site of the dopamine D2receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The high-resolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligand-binding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, I184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs.
Confirmation by DNA analysis that Contarinia maculipennis (Diptera: Cecidomyiidae) is a polyphagous pest of orchids and other unrelated cultivated plants
Authors: Uechi N. , Tokuda M. , Yukawa J. , Kawamura F. , Teramoto K.K. , Harris K.M.
Published: January 2004

The cytochrome oxidase subunit I gene in mitochondrial DNA of 53 larvae of Contarinia maculipennis Felt from flower buds of various host plants, collected from Hawaii, Japan and Thailand was analysed. Monophyly of the clade including C. maculipennis from Hawaii, Thailand and Japan was supported. There was no sequential variation within the specimens from Hawaii and Japan, which differed from one another by 6 bp (1.37%). Three haplotypes were recognized in specimens from Thailand but differences from Hawaiian and Japanese specimens were small. Overall, there were no differences in the 146 deduced amino acid residues. It is therefore concluded that C. maculipennis is a polyphagous species that can develop on plant hosts representing at least seven botanical families. This pest of Dendrobium flower buds in glasshouses is considered to have entered Hawaii, Florida and Japan from Southeast Asia, and was recently intercepted in the Netherlands. Infestations have established and spread in orchid glasshouses, causing concern about the possibility of more extensive damage to orchids and to crops, such as bitter gourd, grown in close proximity to orchid glasshouses in Japan. The potential usefulness of DNA analysis in determining host plant ranges of morphologically identical cecidomyiid species that are currently identified solely on differences of host plant is emphasized.
Amino acid availability: aspects of chemical analysis and bioassay methodology
Authors: Moughan Paul J.
Published: January 2004

It is important to be able to characterise foods and feedstuffs according to their available amino acid contents. This involves being able to determine amino acids chemically and the conduct of bioassays to determine amino acid digestibility and availability. The chemical analysis of amino acids is not straightforward and meticulousness is required to achieve consistent results. In particular and for accuracy, the effect of hydrolysis time needs to be accounted for. Some amino acids (for example, lysine) can undergo chemical modification during the processing and storage of foods, which interferes with amino acid analysis. Furthermore, the modified amino acids may also interfere with the determination of digestibility. A new approach to the determination of available lysine using a modifiedin vivodigestibility assay is discussed. Research is required into other amino acids susceptible to structural damage. There is recent compelling scientific evidence that bacterial activity in the small intestine of animals and man leads to the synthesis and uptake of dietary essential amino acids. This has implications for the accuracy of the ileal-based amino acid digestibility assay and further research is required to determine the extent of this synthesis, the source of nitrogenous material used for the synthesis and the degree of synthesis net of amino acid catabolism. Although there may be potential shortcomings in digestibility assays based on the determination of amino acids remaining undigested at the terminal ileum, there is abundant evidence in simple-stomached animals and growing evidence in human subjects that faecal-based amino acid digestibility coefficients are misleading. Hindgut microbial metabolism significantly alters the undigested dietary amino acid profile. The ileal amino acid digestibility bioassay is expected to be more accurate than its faecal-based counterpart, but correction of the ileal amino acid flow for amino acids of endogenous origin is necessary. Approaches to correcting for the endogenous component are discussed.