bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improving the quality of health information: the contribution of C‐H‐i‐Q
Authors: Hain Tom 
Published: March 2003

AbstractThe quality of information should be the key driver in communicating health messages to the public. Staff at The Centre for Health Information Quality (C‐H‐i‐Q) have worked with an extensive list of partner organizations from a range of disciplines. These stakeholders express a desire to see improvement in the standards of patient information, and display a willingness to embrace new approaches in order to achieve change. The result is an increasingly significant contribution in the development of the information‐exchange model of health‐care, where the aim is to maximize public confidence in health information. Key activities include defining principles of quality assurance, engaging with stakeholders, and developing tools and training for producers and providers of health information.
Regulation of glypican‐1, syndecan‐1 and syndecan‐4 mRNAs expression by follicle‐stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development
Authors: Brucato Sylvie , Bocquet Jean , Villers Corinne 
Published: March 2003

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell‐ to‐cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican‐1, syndecans‐1 and ‐4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle‐stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican‐1 mRNA expression in Sertoli cells from 20‐day‐old rats. On the other hand, syndecan‐1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan‐4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG‐specific and depends on the Sertoli cell developmental stage.
Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids
Authors: Iriko Hideyuki , Nakamura Kazuo , Kojima Hisako , Iida‐Tanaka Naoko , Kasama Takeshi , Kawakami Yasushi , Ishizuka Ineo , Uchida Akihiko , Murata Yoshihiko , Tamai Yoichi 
Published: March 2003

Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai, and their chemical structures were determined. Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion‐exchange chromatography, silicic acid column chromatography, and preparative TLC. The purified GSLs were characterized by methylation analysis, TLC‐immunostaining, liquid secondary ion MS, MALDI‐TOF MS, and 1H‐NMR. Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono‐, di‐, tri‐, tetra‐, and pentasaccharides. The GSL Galβ1–4(Fucα1–3)Glcβ1–3Galβ1‐Cer was found in adult worms but not in plerocercoids, whereas Galβ1–4 (Fucα1–3)Glcβ1–3(Galβ1–6)Galβ1‐Cer was found in both adult worms and plerocercoids. We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei, and termed them ‘spirometosides’[Kawakami, Y. et al. (1996) Eur J. Biochem. 239, 905–911]. The core structure of spirometosides, Galβ1–4Glcβ1–3 Galβ1‐Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms. In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth‐dependent changes in composition were documented. We found a novel dihexosylceramide, Glcβ1–3Galβ1‐Cer, which is a possible precursor for spirometosides. Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host's intestine. Our findings indicate that spirometosides are involved in host–parasite interaction.
Redox‐sensitive loops D and E regulate NADP(H) binding in domain III and domain I–domain III interactions in proton‐translocating Escherichia coli transhydrogenase
Authors: Johansson Carina , Pedersen Anders , Karlsson B. Göran , Rydström Jan 
Published: March 2003

Membrane‐bound transhydrogenases are conformationally driven proton‐pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Δp, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III.With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine‐free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues βA398, βS404, βI406, βG408, βM409 and βV411 in loop D, and residue βY431 in loop E were selected. In addition, the previously made mutants βD392C and βT393C in loop D, and βG430C and βA432C in loop E, were included. All loop D and E mutants, especially βI406C and βG430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild‐type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine‐free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.
Promoter analysis of the human succinate dehydrogenase iron‐protein gene
Authors: Au Harry C. , Scheffler Immo E. 
Published: March 2003

The iron‐sulfur subunit of succinate dehydrogenase is one of the four subunits of complex II of the mitochondrial electron transport chain. Its gene, SDH2, is one of the four nuclear‐encoded genes for this complex. Reporter gene analysis of the human SDH2 promoter indicates that it is transcriptionally regulated by the nuclear respiratory factors NRF‐1 and NRF‐2. Their binding sites reside immediately upstream (within 90 bp) of the transcription start site. Site‐directed mutagenesis of either site lowers the reporter gene activity by tenfold to a basal level. Gel shift experiments and competition experiments with the authentic NRF‐1 and NRF‐2 DNA oligomers from previously characterized nuclear respiratory genes strengthen the proposed role of these two transcriptional regulators. These experiments extend the proposed regulatory role of these two transcription factors to complex II of the respiratory chain.The expression of three of the four genes of complex II was also examined when mouse myoblast C2C12 cells were induced to differentiate into myotubes. Up‐regulation upon differentiation in tissue culture is only modest, 2−3 fold over the myoblast cells.
The prothrombin gene is expressed in the rat kidney
Authors: Grover Phulwinder K. , Dogra Satish C. , Davidson Benjamin P. , Stapleton Alan M. F. , Ryall Rosemary L. 
Published: March 2003

There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney.Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3‐phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis.All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR‐derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.
Cloning, expression and characterization of a Family 48 exocellulase, Cel48A, from Thermobifida fusca
Authors: Irwin Diana C. , Zhang Sheng , Wilson David B. 
Published: March 2003

The gene for a 104‐kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose‐binding domain at the N‐terminus, followed by a FNIII‐like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 µmol cellobiose·min−1·mL−1, more than 15‐fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 °C. Using swissmodel, the amino‐acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.
Nicotinamide adenine dinucleotide species in the horseradish peroxidase–oxidase oscillator
Authors: Kirkor Ewa S. , Scheeline Alexander 
Published: March 2003

NADH chemistry ancillary to the oscillatory peroxidase–oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 ± 0.2) × 105 m−1·s−1 and CpII to HRP at 1 × 105 m−1·s−1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 × 106 m−1·s−1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of α‐NADH, not only the β anomer as previously assumed. Rate constants of α‐ and β‐NADH reactions with CpI are (7.4 ± 0.4) × 105 m−1·s−1, and (1.7 ± 0.2) × 105 m−1·s−1, and with CpII are estimated as 5 × 104 m−1·s−1, and 4 × 104 m−1·s−1.Apparent rate constants of reduction of methylene blue (MB) to leuco‐methylene blue (MBH) are 3.8 × 104 m−1·s−1 for NADH and 6.4 × 104 m−1·s−1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 ± 0.2) × 103 m−1·s−1 All the rates were measured in 0.1 m acetate buffer, pH 5.1.
Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae
Authors: Roethlisberger Peter , Iida‐Tanaka Naoko , Hollemeyer Klaus , Heinzle Elmar , Ishizuka Ineo , Fischer Werner 
Published: March 2003

The Streptococcus sp. studied here is closely related to Streptococcus pneumoniae with 98.6% 16S rRNA similarity and 65% DNA/DNA homology. We isolated the lipoteichoic acid and the membrane glycolipids whose structures were established using conventional procedures and NMR spectroscopy. The lipoteichoic acid contains a linear 1,3‐linked poly(glycerophosphate) chain which is partly substituted with d‐alanine ester and is phosphodiester‐linked to O6 of β‐d‐Galf(1→3)acyl2Gro. This lipoteichoic acid is the first example in which a monohexosylglycerol serves as the glycolipid anchor; and with an average chain length of 10 glycerophosphate residues it is the shortest known to date. MS analysis, applied for the first time to a native acylated lipoteichoic acid, revealed a continuous increase in chain length from seven to 17 glycerophosphate residues with a maximum at 10, and allowed identification of the fatty acid combinations. Membrane glycolipids consisted of β‐d‐Galf(1→3)acyl2Gro (9%), α‐d‐Glcp(1→3)acyl2Gro (22%), α‐d‐Galp(1→2)‐α‐d‐Glcp(1→3)acyl2Gro (64%) and α‐d‐Galp(1→2)‐(6‐O‐acyl)‐α‐d‐Glcp(1→3)acyl2Gro (5%). It is noteworthy that in lipoteichoic acid biosynthesis, Galfacyl2Gro, a less abundant membrane glycolipid, is selected as the lipid anchor. Despite the genetic relatedness to Streptococcus pneumoniae, the lipoteichoic acid structure is quite different to the complex structure of pneumococcal lipoteichoic acid [T. Behr et al. (1992) Eur. J. Biochem.207, 1063–1075], thus providing an example that minor differences in DNA sequence exert major changes in macromolecular structure.
Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies
Authors: Beghetto Claudia , Renken Christian , Eriksson Ove , Jori Giulio , Bernardi Paolo , Ricchelli Fernanda 
Published: March 2003

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein‐stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers.The calcein‐photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500–600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.
Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides
Authors: Guay Jocelyne , Falgueyret Jean‐Pierre , Ducret Axel , Percival M. David , Mancini Joseph A. 
Published: March 2003

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S‐transferase‐fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S‐transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time‐dependent and occurred by an apparent one‐step mechanism. The cathepsin K propeptide had a Ki of 3.6–6.3 nm for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240‐fold selective inhibitor of cathepsin K (Ki = 0.27 nm) and cathepsin L (Ki = 0.12 nm) compared with cathepsin S (Ki = 65 nm). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nm) than cathepsin S (Ki = 7.6 nm) itself or cathepsin K (Ki = 7.0 nm). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J.E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745–750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
Expression and characterization of truncated forms of humanized L243 IgG1
Authors: Lund John , Takahashi Noriko , Popplewell Andrew , Goodall Margaret , Pound John D. , Tyler Ruth , King David J. , Jefferis Royston 
Published: March 2003

The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (α‐MHC Class II) lacking a CH3 domain pair (ΔCH3‐IgG1), single‐chain Fv fusion proteins with Fc or a hinge‐CH2 domain, Fc with/out a hinge, and a single CH2 domain.Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcγ receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30–83%) or sialylated (9–21%) oligosaccharide chains with the highest levels observed for the single CH2 domain. These data show which architectural components influence IgG glycosylation processing and that the (CH3)2 pair is particularly influential.When MHC Class II bearing (JY) cells were sensitized with L243 ΔCH3‐IgG1, scFvFc, or scFvhCH2 they elicited superoxide production, from U937 cells, at levels of 35–45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhCH2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhCH2 homo‐dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcγRI.
Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S
Authors: Hayden Bronagh M. , Engel Paul C. 
Published: March 2003

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD+ but is catalytically inactive. The mutant proteins were denatured separately in 4 m urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mm NAD+, 1 m fluoride and artificial chaperones (4 mm polyoxyethylene 10 lauryl ether and 1.6 mmβ‐cyclodextrin). Under these conditions ≈ 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion‐exchange chromatography. Ellman’s reagent, 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB), which binds close to the NAD+ binding site, thus abolishing coenzyme binding in the wild‐type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye‐ligand affinity chromatography on a Procion Red HE‐3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB‐modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis–Menten kinetics when the NAD+ concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that ‘nonlinear’ behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.