bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Interaction of Bile Acids from Patients with Inborn Errors of Bile Acid Synthesis with Hepatocellular Bile Acid Transporters
Authors: Stieger Bruno , Zhang Jie , O'Neill Brigitte , Sjövall Jan , Meier Peter J. 
Published: July 2004

People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3β‐hydroxy‐Δ5‐C27‐steroid dehydrogenase/isomerase form 3β,7α‐dihydroxy‐and 3β,7α,12α‐trihydroxy‐5‐cholenoic acids, the sulfated and partially glycine‐conjugated forms of which are found in their urine and bile. 3‐Oxo‐Δ4 bile acids are detected in the urine of patients with a deficiency of 5β‐reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients.The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3β,7α‐dihydroxy‐5‐cholenoic acid and 7α‐hydroxy‐3‐oxo‐4‐cholenoic acid both inhibited the apical, ATP‐dependent transport system for cholyltaurine in a competitive manner with Km values of 15 μM and 16 μM, respectively. Radioactively labeled 3β,7α‐dihydroxy‐5‐cholenoyltaurine and 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP‐dependent bile acid transport system, only 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine was a competitive inhibitor of the sodium‐dependent transport system for cholyltaurine with a Km of 16 μM. Studies with radioactively labeled 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine and 3β,7α‐dihydroxy‐5‐cholenoyltaurine revealed that 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine was transported in a sodium‐dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3β,7α‐dihydroxy‐5‐cholenoyltaurine was not transported in a sodium‐dependent way.These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP‐dependent transport system for bile acids which constitutes the rate‐limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium‐dependent and the apical ATP‐dependent transport system for cholyltaurine might have different recognition sites for bile acids.
Model of the pH‐Dependence of the Concentrations of Complexes Involving Metabolites, Haemoglobin and Magnesium Ions in the Human Erythrocyte
Authors: Mulquiney Peter J. , Kuchel Philip W. 
Published: July 2004

The rate of glucose consumption and the concentrations of glycolytic intermediates in human erythrocytes have long been known to be pH sensitive. Despite the extensive literature on modelling erythrocyte metabolism, no model developed so far can adequately describe all of these pH‐dependent changes. None of these models have included all the significant association reactions between metabolites, Hb and Mg2+ that will influence metabolism. As part of a larger enterprise to develop a detailed model of erythrocyte glycolysis, we present a sub‐model which predicts, as a function of pH and oxygenation state, the concentrations of free and Mg2+‐bound metabolites that are substrates, co‐factors and effectors of glycolysis. This model shows that pH changes around physiological values can cause large changes in the distribution of metabolites between free, bound and Mg2+‐complexed forms, based on binding interactions alone; in oxygenated cells, at pH 7.2–7.6, many glycolytic intermediates undergo changes in concentration of 50–100%. The model also predicts intracellular concentrations of free Mg2+ in erythrocytes to be 0.4 mM and 0.64 mM in oxygenated and deoxygenated cells, respectively, assuming a total magnesium concentration of 3 mM (≈88% of the total magnesium usually found in erythrocytes). This is in close agreement with the values found by Flatman [Flatman, P. W. (1980)J. Physiol. 300, 19–30] and the finding by Flatman and Lew [Flatman, P. & Lew, V. L. (1977)Nature 267, 360–362] that the main Mg2+ buffer systems bind ≈90% of Mg2+ in the cell.Hexokinase has a high ‘flux control coefficient’ in human erythrocyte glycolysis, so the dependence of its rate on the pH and oxygenation state of haemoglobin is important. With a low oxygen tension and an intracellular pH of 7.34, the major inhibitor of its activity (2,3‐bisphosphoglycerate) is 85 % bound to either haemoglobin or Mg2+, and the maximum possible flux of substrate via it would be 2.05 mmol L erythrocytes−1 h−1. However, if the haemoglobin were saturated with oxygen, and the pH were 7.2, it was calculated that the maximum rate would be 1.48 mmol L erythrocytes−1 h−1; this is primarily due to a doubling of the free 2,3‐bisphosphoglycerate concentration. However, the full extent of the inhibition is counteracted because the concentration of the Mg2+‐2,3‐bisphosphoglycerate would be approximately doubled. Many other similar comparisons are possible with this new model, which highlights the complex network of interactions between haemoglobin, Mg2+, H+ and the metabolites as substrates and effectors of the glycolytic reactions.
Memory and Imprinting Effects in Multienzyme Complexes
Authors: Avilan Luisana , Gontero Brigitte , Lebreton Sandrine , Ricard Jacques 
Published: July 2004

A bienzyme complex made up of phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde‐3‐phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde‐3‐phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde‐3‐phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two non‐consecutive reactions.The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase.The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells
Authors: Lerche Carole , Fautrel Alain , Shaw Peter M. , Glaise Denise , Ballet Francois , Guillouzo Andre , Corcos Laurent 
Published: July 2004

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P‐450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase α (GSTα), aldehyde dehydrogenase and epoxide hydrolase in co‐cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O‐deethylation activity was well maintained and ethoxyresorufin O‐deethylation activity gradually decreased during co‐culture time. In addition, we showed that phenobarbital and 3‐methylcholanthrene treatments resulted in a significant increase of these activities.Two general patterns of accumulation of liver‐specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GSTa, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6‐fold) and GSTa (3.9‐fold) mRNA expression in 3‐methyl cholanthrene‐treated co‐cultures and induction of CYP2B1/2 (19‐fold), CYP2C6 (10‐fold), CYP3A1/2 (11.2‐fold), GSTα (9‐fold), aldehyde dehydrogenase (6‐fold) and epoxide hydrolase (5‐fold) mRNA expression in phenobarbital‐treated co‐cultures. Furthermore, we demonstrated that liver‐specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co‐culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells.Finally, to investigate whether co‐cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co‐culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163‐bp DNA sequence element reported to confer phenobarbital responsiveness. A 2–3‐fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital‐treated co‐cultures transfected with the phenobarbital‐responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments.Our results demonstrate that the co‐culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
Characterization of The Active Site of Schwanniomyces Occidentalis Glucoamylase by In Vitro Mutagenesis
Authors: Hülseweh Birgit , Dahlems Ulrike M. , Dohmen Jürgen , Strasser Alexander W. M. , Hollenberg Cornelis P. 
Published: July 2004

Site‐directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase. The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined. Mutants were transcribed and translated similar to the wild‐type glucoamylase. Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions.Asp470 was shown to be essential for the enzyme activity. Replacement of Asp470 by glycine led to a complete loss of activity. We suppose that Asp470 serves as a general acid‐base and stabilizes the formation of the intermediate carbenium ion. Substitution of Trp468 by alanine affected predominantly the α‐1,6 activity and not the α‐1,4 activity of the enzyme. The exchange impaired substrate binding as well as enzymatic catalysis. An influence of amino acid 474 on the substrate specificity could not be demonstrated. Exchanges at position 474 exhibited Kn, and Vmax values similar to wild‐type glucoamylase.
Role of the Stimulatory Proteins Sp1 and Sp3 in the Regulation of Transcription of the Rat Pyruvate Kinase M Gene
Authors: Netzker Roland , Weigert Cora , Brand Karl 
Published: July 2004

Site‐directed mutagenesis of cis‐regulatory elements in the 5′ flanking region of the rat pyruvate kinase M gene revealed that two out of the three GC boxes (–1337–124 and–48/–39) are involved in the stimulation of a core promoter (–35/+46). These two regions were also protected in DNasel foot‐printing assays. Sp1 and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments.Cotransfections in Drosophila SL2 cells revealed a strong stimulatory function of Sp1 and a synergistic effect of Sp3 to Sp1 in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected.These data indicate that binding of Sp1 at two GC boxes is required for full promoter activity of the pyruvate kinase M gene and thus contributes to the observed cell‐cycle‐dependent expression of this enzyme in proliferating rat thymocytes.
Evolution of The Aminoacyl‐tRNa Synthetase Family and The Organization of the Drosophila Glutamyl‐Prolyl‐tRNA Synthetase Gene — Intron/Exon Structure of the Gene, Control of Expression of the Two mRNAs, Selective Advantage of the Multienzyme Complex
Authors: Cerini Claire , Semeriva Michel , Gratecos Danielle 
Published: July 2004

In Drosophila, glutamyl‐prolyl‐tRNA synthetase is a multifunctional synthetase encoded by a unique gene and composed of three domains: the amino‐ and carboxy‐terminal domains catalyze the aminoacylation of glutamic acid and proline tRNA species, respectively, and the central domain is made of 75 amino acids repeated six times amongst which 46 are highly conserved and constitute the repeated motifs [Cerini, C., Kerjan, P., Astier, M., Gratecos, D., Mirande, M. & Sémériva, M. (1991) EMBO J. 10, 4267–4277].The intron/exon organization of the Drosophila gene reveals the presence of six exons among which four are in the 5′‐end encoding glutamic acid activity. Only one exon encodes the repeated motifs. A comparison of introns positions, intron classes and intron/exon boundaries in the Drosophila gene and in its human counterpart is compatible with the intron‐early hypothesis presiding, at least in part, to the evolution of the synthetases.The full‐length fly protein is encoded by a 6.1‐kb mRNA which is expressed throughout development. In addition, a shorter transcript encompasses the 3′‐end of the cDNA and it is especially abundant in 5–10‐h embryos until the first larval stage. Expression of these two mRNAs seems to be controlled by two independent promoters. The 6.1‐kb mRNA promoter is probably localized in the 5′‐end of the gene. The small mRNA promoter resides in the 4th intron and evidence is provided that the mRNA encodes only the domain corresponding to prolyl‐tRNA synthetase and is functional in vivo.Finally, transgenic flies have been established by using constructs corresponding to the three domains of the protein. Overexpression of the repeated motifs leads to a sterility of the flies that suggests a role of these motifs in linking the multienzyme complex to an, as yet, unknown structure of the protein synthesis apparatus.
A Potassium‐Channel Toxin From the Sea Anemone Bunodosoma Granulifera, An Inhibitor for Kv1 Channels — Revision of the Amino Acid Sequence, Disulfide‐Bridge Assignment, Chemical Synthesis, and Biological Activity
Authors: Cotton Jöel , Crest Marcel , Bouet Françoise , Alessandri Nicole , Gola Maurice , Forest Eric , Karlsson Evert , Castaneda Olga , Harvey Alan L. , Vita Claudio , Ménez Andre 
Published: July 2004

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37‐amino‐acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C‐terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C.The toxin BgK was synthesized according according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2‐Cys37, Cys11‐Cys30 and Cys20‐Cys34. The toxin contains a C‐terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C‐terminus in either the carboxylate or carboxamido form.Synthetic BgK inhibits binding of 125I‐α‐dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi‐K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage‐dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker‐related Kv1 channels are equally affected by BgK, while the Shaw‐related channel Kv3.1 is insensitive up to 0.125 pM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd= 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker‐related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ.The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demostrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.
Detection of Metal Binding to Bovine Inositol Monophosphatase by Changes in the Near and Far Ultraviolet Regions of the CD Spectrum
Authors: Rees‐Milton Karen , Thorne Mark , Greasley Peter , Churchich Jorge , Gore Michael G. 
Published: July 2004

Mg2+ ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near‐ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant (Kd) around 275 μM, 292 μM and 302 μM for the wild‐type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219.The addition of Mg2+ also causes a decrease in ellipticity over most of the far‐ultraviolet region of the spectrum (between 205–240 nm). The Kd values describing the binding of Mg2+ ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild‐type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12% change in ellipticity. In the additional presence of 10 mM Pi there is a fourfold increase in the affinity of wild‐type enzyme for Mg2+.It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high‐affinity metal‐binding site (site 1). The spectral changes around 225 nm are associated with interactions at a lower‐affinity site normally occupied by the Mg2+ ion which is reflected by the Km value for this metal ion.Other metal ions such as Ca2+ and Tb3+ (but not Mn2+ or Zn2+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg2+ or Tb3+ (25%) ions than for Ca2+ (8.5%), although two Ca2+‐binding sites with Kd values of 20 μM and 100 μM have been identified.The uncompetitive inhibitor Li+ causes little change in the near‐ultraviolet spectrum in the absence or presence of either substrate or Pi. However, in contrast to other metal ions, Li+ ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.
Investigation of Different Recombinant Isoforms of Grass Group‐V Allergens (Timothy Grass Pollen) Isolated by Low‐Stringency cDNA Hybridization — Antibody Binding Capacity and Allergenic Activity
Authors: Gehlhar Kirsten , Petersen Arnd , Schramm Gabriele , Becker Wolf‐Meinhard , Schlaak Max , Bufe Albrecht 
Published: July 2004

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group‐V allergens by low stringency hybridization with a Phi p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group‐V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group‐V allergens was shown to be in the same range for both subgroups.Analysis of allergenic B‐cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine‐release and skin‐prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group‐V isoallergens in grass pollen, although they can be structurally different, induce a similar B‐cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type‐I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.
Some DNA Targets of the Yeast CYP1 Transcriptional Activator are Functionally Asymmetric
Authors: Naït‐Kaoudjt Rachida , Williams Roy , Guiard Bernard , Gervais Michel 
Published: July 2004

CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA‐binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild‐type CYP1 target sites.In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1‐A/CYCI, UAS1‐A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1‐(1‐ 200)‐peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding, (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1‐(1 ‐ 200)‐peptide is usually a monomer in solution but binds DNA as a dimer.Finally, we found evidence for the presence of two half‐sites with different measured affinities in the asymmetric sequences of some CYP1 targets.