bluuebunny/crossref_metadata_2025_split huggingface dataset:
Whose priorities count? Comparison of community‐identified health problems and Burden‐of‐Disease‐assessed health priorities in a district in Uganda
Authors: Kapiriri Lydia , Frithjof NorheimMD PhD Ole 
Published: March 2003

Objectives The aim of the study was to compare health problems as defined quantitatively by the Burden of Disease study to those defined by the community. The secondary aim was to explore the potential for using qualitative participatory methodologies as tools for planing and priority setting.Design Interviews and group discussions with a purposely sampled set of community members (n=51) and community leaders (n=6). The Nominal group technique, as well as in‐depth interviews, were used to identify major health problems – as perceived by the community. Epidemiological data on the major health problems were derived from the national Burden of Disease study.Results Community perceived health problems were similar to those identified by the burden of disease study. Reasons given for the ranking included prevalence, fatality, social and cultural stigma. Social stigma and cultural values were not considered in the burden of disease studies. However, socially stigmatized diseases were considered to be more serious compared to non‐stigmatised conditions, in spite of their low prevalence. Poverty and lack of knowledge were the perceived major causes of ill‐health in the community.Conclusions Qualitative approaches like the nominal group technique may be useful in eliciting community values that could supplement quantitative information like that elicited by the Burden of Disease study. Such a mixed approach would capture both epidemiologicaly assessed and community felt needs in the priority setting process.
Contact allergy to fragrances: clinical and experimental investigations of the fragrance mix and its ingredients
Authors: Duus Johansen Jeanne 
Published: March 2003

Denne afhandling er af Det Sundhedsvidenskabelige Fakultet ved Københavns Universitet antaget til offentligt at forsvares for den medicinske doktorgrad.København den 24. oktober 2001Hans Hultborndekan
Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases
Authors: Grobe Kay , Pöppelmann Marco , Becker Wolf‐Meinhard , Petersen Arnd 
Published: March 2003

Expansins are a family of proteins that catalyze pH‐dependent long‐term extension of isolated plant cell walls. They are divided into two groups, α and β, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall.Our group reported papain‐like properties of β‐expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin‐mediated wall extension. Here, we report additional data showing that β‐expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N‐glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full‐length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between β‐expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino‐acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site‐directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of β‐expansins. These results indicate a common origin of cathepsin B and β‐expansins, especially if taken together with their previously known biochemical properties.
Chimeric receptor analyses of the interactions of the ectodomains of ErbB‐1 with epidermal growth factor and of those of ErbB‐4 with neuregulin
Authors: Kim Jae‐Hoon , Saito Kazuki , Yokoyama Shigeyuki 
Published: March 2003

A series of chimeric receptors was generated between the epidermal growth factor (EGF) receptor, ErbB‐1, and its homologue, ErbB‐4, to investigate the roles of the extracellular domains (I–IV) in the ligand specificities. As compared with ErbB‐1 and the chimeras with both domains I and III of ErbB‐1, the chimeras with only one of these domains exhibited reduced binding of 125I‐labeled EGF. Particularly, the contribution of domain III was appreciably larger than that of domain I of ErbB‐1 in 125I‐labeled EGF binding. Nevertheless, the chimeras with domain III of ErbB‐1 and domain I of ErbB‐4 were prevented from binding to	125I‐labeled EGF competitively by the ErbB‐4 ligand, neuregulin (NRG). On the other hand, NRG did not compete with 125I‐labeled EGF for binding to the chimeras with the ErbB‐1 domain I and the ErbB‐4 domain III. Therefore, NRG binding to ErbB‐4 depends much more on domain I than on domain III. With respect to autophosphorylation and subsequent ERK activation, EGF activated the chimeras with either domain I or III of ErbB‐1. In contrast, NRG activated the chimeras with the ErbB‐4 domain I and the ErbB‐1 domain III, but not those with the ErbB‐1 domain␣I and the ErbB‐4 domain III. Therefore, the relative contributions between domains I and III of ErbB‐4 in the NRG signaling are different from those of ErbB‐1 in the EGF signaling.
Soluble silk‐like organic matrix in the nacreous layer of the bivalve Pinctada maxima
Authors: Pereira‐Mouriès Lucilia , Almeida Maria‐José , Ribeiro Cristina , Peduzzi Jean , Barthélemy Michel , Milet Christian , Lopez Evelyne 
Published: March 2003

Nacre organic matrix has been conventionally classified as both ‘water‐soluble’ and ‘water‐insoluble’, based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid‐rich, silk‐fibroin‐like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra‐pure water, displays many of the characteristics of what until now has been called ‘insoluble matrix’. We present the results obtained on this extract and on a conventional EDTA‐soluble matrix, with various characterization methods: fractionation by size‐exclusion and anion‐exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara [In: Biomineralization and Biological Metal Accumulation, Westbroek, P. & deJong, E.W., eds, (1983) pp. 225–230. Reidel, Dordrecht, Holland] and Weiner and Traub [Phil. Trans. R. Soc. Lond. B (1984) 304, 425–434] may no longer be valid. The most recent model, proposed by Levi‐Kalisman et al. [J. Struct. Biol. (2001) 135, 8–17], seemed to be more in accordance with our findings.
Probing intermolecular protein–protein interactions in the calcium‐sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)
Authors: Jensen Anders A. , Hansen Jakob L. , Sheikh Søren P. , Bräuner‐Osborne Hans 
Published: March 2003

The calcium‐sensing receptor (CaR) belongs to family C of the G‐protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non‐covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C receptors takes place at the ATD and that this causes the ATD dimer to twist. However, very little is known about the translation of the ATD dimer twist into G‐protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers.In this study we have attempted to delineate the agonist‐induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations. Stable and highly receptor‐specific BRET signals were obtained in tsA cells transfected with Rluc‐ and GFP2‐tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least parts of the two 7TMs of the CaR homodimer are in close proximity in the inactivated state of the receptor and do not move much relative to one another upon agonist activation. However, we cannot exclude the possibility that the BRET technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.
Demonstration of leukotriene‐C4 synthase in platelets and species distribution of the enzyme activity
Authors: Tornhamre Susanne , Sjölinder Mikael , Lindberg Åsa , Ericsson Inger , Näsman‐Glaser Barbro , Griffiths William J. , Lindgren Jan Åke 
Published: March 2003

Human platelets have been demonstrated to possess leukotriene (LT)‐C4 synthase activity and may thus be involved in transcellular 5(S)‐hydroxy‐6(R)‐S‐glutathionyl‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acid (LTC4) synthesis. In this study, platelets from seven different species were screened for LTC4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70 % conversion of 5(S)‐trans‐5,6‐oxido‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acid (LTA4) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC4 after incubation with LTA4. The results demonstrate that LTC4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced 5(S),12(R)‐dihydroxy‐6,14‐cis‐8,10‐trans‐eicosatetraenoic acid (LTB4), indicating the presence of LTA4 hydrolase in porcine platelets.A protein with a molecular mass of approximately 18 kDa and LTC4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N‐terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed Km (for LTA4) and Vmax values of 3.3 μM and 521 nmol mg protein−1 min−1, respectively. In addition, the presence of a mRNA transcript encoding LTC4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N‐terminus demonstrated very high identity between equine and human leukotriene‐C4 synthase in this region.In summary, the present study establishes that platelets contain LTC4 synthase and indicates that this enzyme is widely distributed among platelets from various species.
Studies on the effect of fucosylated and non‐fucosylated finger/growth‐factor constructs on the clearance of tissue‐type plasminogen activator mediated by the low‐density‐lipoprotein‐receptor‐related protein
Authors: Camani Chantal , Gavin Olivier , Bertossa Carole , Samatani Emmanuel , Kruithof Egbert K. O. 
Published: March 2003

Previously we observed that the finger/growth factor (FG) region of tissue‐type plasminogen activator (t‐PA) blocked the low‐density‐lipoprotein‐receptor‐related protein (LRP)‐mediated clearance of t‐PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t‐PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t‐PA by human hepatoma cells. At position 83 it had a serine, whereas human t‐PA has a free cysteine and rodent t‐PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2‐Fc). Two variants were studied, one having Ser83, the other Arg83.The two fucosylated FG2‐Fc chimeras were compared with each other, with non‐fucosylated FG and with intact t‐PA with regard to their effect on the clearance of t‐PA and t‐PA plasminogen‐activator inhibitor type 1 (PAI‐1). For this comparison, LRP‐specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF‐1) the clearance of t‐PA and of t‐PA PAI‐1 was inhibited more than 95 % by receptor‐associated protein, an inhibitor of LRP‐mediated clearance, whereas no t‐PA or t‐PA PAI‐1 clearance was observed in LRP‐deficient PEA‐13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2‐Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t‐PA and t‐PA PAI‐1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non‐fucosylated FG (IC50 1950 nM and 1560 nM) and 75‐fold lower than that of intact t‐PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP‐mediated clearance of t‐PA.
Characterisation of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A and its N‐glycans
Authors: Altmann Friedrich , Paschinger Katharine , Dalik Thomas , Vorauer Karola 
Published: March 2003

Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two‐dimensional electrophoresis. Peaks corresponding to molecular masses of 54.2, 21.2 and 75.5 kDa were observed with matrix‐assisted laser‐desorption/ionization mass spectrometry. The N‐terminal sequences of the larger and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFPP, respectively. Both polypeptides reacted with concanavalin A, indicating their glycoprotein nature.Upon digestion of PNGase with pepsin, the N‐linked oligosaccharides were released with active PNGase and anlysed as their 2‐aminopyridine derivatives by two‐dimensional HPLC and by matrix‐assisted laser‐desorption mass spectrometry. The most abundant N‐glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and α1,3‐linked fucose. The other structures either had an additional mannose residue and/or lacked the fucose.PNGase A was largely but not absolutely resistant to self‐deglycosylation. However, only at an extremely high enzyme/substrate ratio, N‐glycans released from PNGase A itself caused a detectable contamination of a PNGase digest of a glycopeptide.
Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP‐dependent protein kinase, Tpk1 and Tpk2
Authors: Zähringer Harald , Holzer Helmut , Nwaka Solomon 
Published: March 2003

In Saccharomyces cerevisiae cAMP‐dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway. Target enzymes, e.g. neutral trehalase, are activated or inactivated rapidly by cAPK‐mediated phosphorylation. In addition, stress‐induced transcription of genes of the general stress‐response, e.g. HSP12, is negatively regulated via cAPK.We have investigated the effect of low cAPK activity on the stress‐induced expression of neutral trehalase Nth1p. For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK. It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat‐stress‐induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK11tpk2tpk3 mutants and wild‐type cells. However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant. Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat‐induced increase of neutral trehalase activity. In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p. Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S. cerevisiae. Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p. Stabilization and activation of neutral trehalase may be regulated by these sites. Furthermore, our data suggest that the heat‐stress‐induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non‐detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat‐shock‐recovery defect reported for the nth1Δ mutant.
Transferrins
Authors: Bou Abdallah Fadi , El Hage Chahine Jean‐Michel 
Published: March 2003

Fe(III) uptake by the iron‐delivery and iron‐scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo‐ovotransferrin loses two protons with K1a = 50 ± 1 nM and K2a = 4.0 ± 0.1 nM. These acid‐base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 ± 0.15 mM and KN = 110 ± 10 mM. FeNAc3 exchanges its Fe(III) with the C‐site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 ± 25 M−1 s−1, reverse rate constant k−1 = (6.0 ± 0.1)×103 M−1 s−1 and equilibrium constant K1 = 0.11 ± 0.01. This iron‐protein intermediate loses then a single proton, K3a = 3.50 ± 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 ± 0.01 s−1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 ± 0.05)×10−2 s−1. These modifications in the monoferric protein conformation are essential for iron uptake by the N‐site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15 000 s.The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics ?
Biochemical characterization, molecular cloning and expression of laccases – a divergent gene family – in poplar
Authors: Ranocha Philippe , McDougall Gordon , Hawkins Simon , Sterjiades Raja , Borderies Gisèle , Stewart Derek , Cabanes‐Macheteau Marion , Boudet Alain‐M. , Goffner Deborah 
Published: March 2003

The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2‐independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90‐kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm. The cDNA clones corresponding to the 90‐kDa and 110‐kDa proteins, lac90 and lac110, were obtained by a PCR‐based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene‐specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
Protection of mice against a lethal influenza virus challenge after immunization with yeast‐derived secreted influenza virus hemagglutinin
Authors: Saelens Xavier , Vanlandschoot Peter , Martinet Wim , Maras Marleen , Neirynck Sabine , Contreras Roland , Fiers Walter , Jou Willy Min 
Published: March 2003

The A/Victoria/3/75 (H3N2‐subtype ) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C‐terminal transmembrane anchor‐coding sequence was fused to the Saccharomyces cerevisiaeα‐mating factor secretion signal and placed under control of the methanol‐inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol‐containing medium resulted in the secretion of recombinant non‐cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA‐conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N‐linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10–12 glycosyl residues.  Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse‐adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2‐subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.