bluuebunny/crossref_metadata_2025_split huggingface dataset:
A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module
Authors: Kroon Paul A. , Williamson Gary , Fish Neville M. , Archer David B. , Belshaw Nigel J. 
Published: August 2003

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino‐acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120‐bp ORF, faeB, was obtained which encoded a putative 353‐residue preprotein including an 18‐residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N‐terminal domain comprised 276 amino acids, contained a G‐X‐S‐X‐G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3‐hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C‐terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate‐binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.
Interplay between repressing and activating domains defines the transcriptional activity of IRF‐1
Authors: Kirchhoff Sabine , Oumard André , Nourbakhsh Mahtab , Levi Ben‐Zion , Hauser Hansjörg 
Published: August 2003

Interferon regulatory factor‐1 (IRF‐1) is a transcriptional activator with weak activation capacity. By defining the transcriptional activation domain of IRF‐1 we identified two activator fragments located between amino acids 185 and 256 functioning in an additive manner. Another fragment of IRF‐1, which has no activator function alone, acts as a strong enhancer element of these activator sequences. This enhancer element resides between the activator domains and the C‐terminus. In addition, we identified a novel type of inhibitory domain in the N‐terminal 60 amino acids of IRF‐1 which strongly inhibits its transcriptional activity. Because this fragment is conserved in all interferon regulatory factors, we found similar repression effects in the corresponding fragments in IRF‐2, IRF‐3 and interferon consensus sequence binding protein (ICSBP/IRF‐8). Interestingly, the corresponding sequence in p48/IRF‐9 is divergent, so that it does not show this inhibitory activity. A five‐amino‐acid sequence distinguishes the p48/IRF‐9 N‐terminus from the homologous parts in other interferon regulatory factors containing the repressing function. Replacing the diverged amino acids in IRF‐1 with the corresponding sequence of p48/IRF‐9 resulted in a loss of inhibitory activity within IRF‐1. The opposing activities within interferon regulatory factors may contribute to balanced or tuned regulation of gene activation, depending on the promoter context.
Mechanisms of differential transferrin receptor expression in normal hematopoiesis
Authors: Sposi Nadia M. , Cianetti Luciano , Tritarelli Elena , Pelosi Elvira , Militi Stefania , Barberi Tiziano , Gabbianelli Marco , Saulle Ernestina , Kühn Lukas , Peschle Cesare , Testa Ugo 
Published: August 2003

We have investigated the expression of transferrin receptor (TfR) iron regulatory protein‐1 (IRP‐1) and iron regulatory protein‐2 (IRP‐2) in liquid suspension culture of purified hematopoietic progenitor cells (HPCs) induced by a growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid, granulocytic, monocytic and megakaryocytic lineages.In initial HPC differentiation, TfR expression is induced in both erythroid and granulopoietic cultures. In late HPC differentiation (i.e. starting from day 5 of culture) and then differentiated precursor maturation, the TfR gene is highly expressed in the erythroid lineage, whereas it is sharply downmodulated in the granulopoietic, monocytopoietic and megakaryocytic series. The elevated TfR expression in erythroid cells is: (a) mediated through a high rate of TfR gene transcription; (b) modulated by intracellular iron levels; (c) mediated by TfR mRNA stabilization through the iron regulatory protein (IRP), in that IRP‐1 activity is high in erythroid lineage as compared to the levels observed in other hemopoietic lineages; and (d) dependent on exogenous erythropoietin (Epo) (this is indicated by the marked TfR and IRP‐1/IRP‐2 downmodulation after Epo starvation).Interestingly, analysis of IRP‐1 and IRP‐2 expression during hemopoietic differentiation showed that: (a) IRP‐1 expression was maintained during all steps of erythroid differentiation, while it was lost in the other hemopoietic lineages; (b) IRP‐2 expression was observed during all stages of hemopoietic differentiation in all four lineages. However, IRP‐1 and IRP‐2 expression and activity are induced when monocytes, which express only low levels of IRP‐1 and IRP‐2, are induced to maturation to macrophages.These studies indicate that: (a) in normal erythropoiesis, the hyperexpression of TfR, starting from early erythroid HPC differentiation, is Epo‐dependent and mediated via transcriptional and post‐transcriptional mechanisms; (b) in the granulopoietic, monocytopoietic and megakaryocytic pathways, the TfR is first induced and then downmodulated (the latter phenomenon is mediated via transcriptional suppression of the TfR gene and IRP inactivation).
Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana
Authors: Eeckhout Dominique , Fiers Esbjörn , Sienaert Rebecca , Snoeck Veerle , Depicker Ann , De Jaeger Geert 
Published: August 2003

In order to obtain recombinant antibody fragments that bind the cell‐cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single‐chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N‐terminally fused to a His6‐tag (His‐CDC2aAt) and the other was made out of an anti‐PSTAIRE hybridoma cell line. Six specific His‐CDC2aAt‐binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His‐CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen‐binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two‐hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen‐binding activity.
Muscarinic toxin‐like proteins from cobra venom
Authors: Kukhtina Viktoria V. , Weise Christoph , Muranova Tatyana A. , Starkov Vladislav G. , Franke Peter , Hucho Ferdinand , Wnendt Stephan , Gillen Clemens , Tsetlin Victor I. , Utkin Yuri N. 
Published: August 2003

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino‐acid sequences determined. They consist of 65‐amino‐acid residues and have four disulfide bridges. A comparison of the amino‐acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP‐1 and MTLP‐2) share a high degree of similarity (55–74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP‐3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide‐confined loops, but shows less similarity to these toxins (30–34% sequence identity). It is almost identical with a neurotoxin‐like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only.The binding affinities of the isolated muscarinic toxin‐like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1–m5) was determined in competition experiments with N‐[3H]methylscopolamine using membrane preparations from CHO‐K1 cells, which express these receptors. We found that MTLP‐1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 µm was determined. MTLP‐2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 µm. MTLP‐1 showed no inhibitory effect on α‐cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 µm.
Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics
Authors: Smith Leif , Novák Jan , Rocca Jim , McClung Scott , Hillman J. D. , Edison Arthur S. 
Published: August 2003

The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether‐containing residues from the dehydrated residues. This double‐labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double‐labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized.
Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobeBilophila wadsworthia
Authors: Laue Heike , Cook Alasdair M. 
Published: August 2003

Bilophila wadsworthiaRZATAU is a Gram‐negative bacterium which converts the sulfonate taurine (2‐aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72‐fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5′‐phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5′‐phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were:Km = 7.1 mm,Vmax = 1.20 nmol·s−1, and for pyruvate:Km = 0.82 mm,Vmax = 0.17 nmol·s−1. The purified enzyme was able to transaminate hypotaurine (2‐aminosulfinate), taurine, β‐alanine and with low activity cysteine and 3‐aminopropanesulfonate. In addition to pyruvate, 2‐ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50‐kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase fromBacillus subtilis.
Purification and properties of hydroquinone hydroxylase, a FAD‐dependent monooxygenase involved in the catabolism of 4‐hydroxybenzoate in Candida parapsilosis CBS604
Authors: Eppink Michel H. M. , Cammaart Erwin , van Wassenaar Dick , Middelhoven Wouter J. , van Berkel Willem J. H. 
Published: August 2003

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4‐hydroxybenzoate through the initial formation of hydroquinone (1,4‐dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4‐hydroxybenzoate, 2,4‐dihydroxybenzoate, 1,3‐dihydroxybenzene or 1,4‐dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76‐kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD.Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho‐hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 °C, this step does not limit the rate of overall catalysis.During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N‐Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.
Conversion between two cytochalasin B‐binding states of the human GLUT1 glucose transporter
Authors: Gottschalk Ingo , Lundqvist Andreas , Zeng Cheng‐Ming , Hägglund Christine Lagerquist , Zuo Shu‐Sheng , Brekkan Eggert , Eaker David , Lundahl Per 
Published: August 2003

Two cytochalasin B‐binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B‐binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton‐depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin‐derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran‐grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size‐exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non‐ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B‐binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran‐grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.
Efficient DNA base excision repair in ataxia telangiectasia cells
Authors: Cappelli Enrico , Rossi Ottavio , Chessa Luciana , Frosina Guido 
Published: August 2003

Ataxia telangiectasia (A‐T) cells are sensitive to a broad range of free‐radical‐producing and alkylating agents. Damage caused by such agents is in part repaired by base excision [base excision repair (BER)]. Two BER pathways have been demonstrated in mammalian cells: a single‐nucleotide‐insertion pathway and a long‐patch pathway involving resynthesis of 2–10 nucleotides. Although early studies failed to detect DNA‐repair defects in A‐T cells exposed to ionizing radiation and radiomimetic agents, more recent experiments performed in non‐dividing A‐T cells and the demonstrated interaction of the A‐T‐mutated protein (ATM) with the BRCA1 gene product suggest that a DNA‐repair defect may underlie, at least in part, the radiation sensitivity in A‐T cells. We have analysed BER of a single abasic site or a single uracil in two A‐T families, using an in vitro BER system. In both families, the mutation involved was homozygous and completely inactivated the ATM protein. No difference was observed between affected individuals and heterozygous or homozygous wild‐type relatives in their capacity to perform DNA repair by either one‐nucleotide insertion or the long‐patch pathway. Hence, the putative DNA‐repair defect in A‐T cells, if any, does not involve BER.
Structural and serological studies of the related O‐specific polysaccharides of Proteus vulgaris O21 and Proteus mirabilis O48 having oligosaccharide‐phosphate repeating units
Authors: Bartodziejska Beata , Toukach Filip V. , Vinogradov Evgeny V. , Senchenkova Sofia N. , Shashkov Alexander S. , Ziolkowski Andrzej , Czaja Jolanta , Perry Malcolm B. , Knirel Yuriy A. , Rozalski Antoni 
Published: August 2003

The O‐specific polysaccharide chains (O‐antigens) of the lipopolysaccharides (LPSs) of Proteus mirabilis O48 and Proteus vulgaris O21 were found to have tetrasaccharide and pentasaccharide repeating units, respectively, interlinked by a glycosidic phosphate. Polysaccharides and an oligosaccharide were derived from the LPSs by various degradation procedures and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H‐detected 1H,13C and 1H,31P HMQC experiments. The following related structures of the repeating units of the O‐antigens were established (top: Proteus mirabilis O48; bottom: Proteus vulgaris O21)The O‐specific polysaccharide of P. vulgaris O21 has the same structure as that of Hafnia allvei 744 and PCM 1194 [Petersson C., Jachymek, W., Klonowska, A., Lugowski, C., Niedziela, T. & Kenne, L. (1997) Eur. J. Biochem., 245, 668–675], except that the GlcN residue carries the N‐acetyl rather than the N‐[(R)‐3‐hydroxybutyryl] group. Serological investigations confirmed the close relatedness of the Proteus and Hafnia O‐antigens studied.
Molecular cloning of NADPH‐cytochrome P450 oxidoreductase from silkworm eggs
Authors: Horike Nanao , Takemori Hiroshi , Nonaka Yasuki , Sonobe Haruyuki , Okamoto Mitsuhiro 
Published: August 2003

Using RT‐PCR, a cDNA fragment of NADPH‐cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three‐day‐old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full‐length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino‐acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S‐transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH‐dependent cytochrome c‐reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21‐hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH‐cytochrome P450 oxidoreductase. Ecdysone 20‐hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three‐day‐old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20‐hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20‐hydroxylase P450.
Tolerability of topiramate in children and adolescents
Authors: Reith D , Burke C , Appleton DB , Wallace G , Pelekanos J 
Published: August 2003

Objective:  To examine the tolerability of topiramate (TPM) in paediatric practice.Methodology:  A retrospective cohort study of patients aged less than 18 years commenced on TPM by paediatric neurologists. Patients were identified from the dispensing databases of two paediatric tertiary referral hospitals and from the authority prescription records of four paediatric neurologists. The clinical data were obtained from the patients’ medical records.Results:  There were 159 patients who were identified as having been commenced on TPM and follow‐up data were available for 127 (80%) patients. The median (range) age at commencement of TPM was 8.1 (0.5−17.9) years, with 12 patients aged less than 2 years. After 4 years, 60% of patients had ceased the medication. Treatment limiting adverse effects included aggression/psychosis (n = 10), cognitive impairment/sedation (n = 6), anorexia/weight loss (n = 4) and desquamation (n = 1). Prior aggression (hazard ratio 5.91 (2.12−16.44)) and female gender (hazard ratio 2.94 (1.02−8.41)) were risk factors for ceasing TPM because of an adverse event. Thirty percent of children commenced on TPM experienced a treatment limiting adverse effect within 2 years of commencement.Conclusions:  The frequency of treatment limiting adverse events in children receiving topiramate is higher than previously reported.
Unintentional residential child injury surveillance in Hong Kong
Authors: Chan CC , Luis Bpk , Chow Cb , Cheng JCY , Wong Tw , Chan K 
Published: August 2003

Objectives:  To provide an overall pattern of morbidity in unintentional residential childhood injuries (URCI) in Hong Kong.Methodology:  A cross‐sectional telephone survey of caregivers of children aged under 16‐years and adolescents suffering from URCI and admitted to three selected local Accident and Emergency Departments.Results:  Falls, cuts and scalds were the most common external causes of URCI observed, while boys predominated in the sample population. Most of the observed URCI were of moderate to mild severity. Children of new immigrant mothers were more likely to receive first aid immediately after the incidents. Parents were aware of potentially injurious behaviour and intervened on occasion, but most resorted to verbal warnings only.Conclusions:  Prevalence of falls among observed URCI offers evidence in support of the hypothesis that the high population density in Hong Kong plays an integral role in understanding mechanisms of morbidity. Parents show concern about URCI but often lack substantial action that modifies injury risk. Considering the local injury differentials, an active prevention effort such as behavioural intervention and education for parents may be useful.