bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidoreduction of butanol in deermice (Peromyscus maniculatus) lacking hepatic cytosolic alcohol dehydrogenase
Authors: CRONHOLM Tomas , NORSTEN‐HÖÖG Carina , EKSTRÖM Gunilla , HANDLER Jeffrey A. , THURMAN Ronald G. , INGELMAN‐SUNDBERG Magnus 
Published: March 2005

In view of conflicting information in the literature regarding enzyme systems responsible for alcohol oxidation in deermice previously reported to lack hepatic alcohol dehydrogenase (ADH) activity, the reversibility of butanol oxidation was studied in vivo and in liver‐perfusion systems. Mixtures of [1,1‐2H2]ethanol and butanol were given intraperitoneally to deermice lacking (ADH−) or possessing (ADH+) ADH activity, followed by analysis of alcohols in blood by GC/MS. 2H exchange between the two alcohols was seen in all experiments. In ADH− deermice, the 2H excess of butanol increased steadily and reached 18 ± 5% after 2.5 h. In ADH+ deermice, butanol was rapidly eliminated and the 2H excess was about 7% after 0.5 h. In similar experiments with rats, the 2H excess was about 40% for 2 h.Perfusions of livers from ADH− deermice with mixtures of unlabelled and 1‐[2H]butanol showed significant but slow intermolecular hydrogen transfer at C1, indicating oxidoreduction catalyzed by a dehydrogenase. Slow reduction of butanal was observed in mitochondria from ADH− deermice. ADH activity with a pH optimum of 10 and Km for ethanol of 6 mM was detected in the inner mitochondrial membranes from rats and deermice. However, low rates of oxidation observed in experiments carried out with perfused livers and in vitro suggest that this enzyme system does not contribute significantly to alcohol oxidation in vivo. Thus, perfused liver from ADH− deermice appears to be a useful system for studies of ADH‐independent oxidation of alcohols. The 2H exchange between the alcohols seen in vivo indicates that both ethanol and butanol are substrates for a common extrahepatic dehydrogenase in ADH− deermice.
Mechanism of the stimulatory effect of a potassium‐rich medium on the phosphorylation of glucose in isolated rat hepatocytes
Authors: VAN SCHAFTINGEN Emile , VANDERCAMMEN Annick 
Published: March 2005

We have investigated the mechanism by which the replacement of a Na+‐rich medium by a K+‐rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603–611].The stimulatory effect of a K+‐rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2‐3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K+‐rich medium was neither due to the formation of fructose 1‐phosphate, nor to changes in the concentrations of fructose 6‐phosphate or Pi, two other effectors of the regulatory protein.Replacement of Na+ by K+ in the medium resulted in a time‐dependent and dose‐dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl− concentration about threefold higher (94 versus 29 meq/l). The effects of the K+‐rich medium on cell volume, Cl− concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45–50 min in the K+‐rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl− concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K+‐rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein.From these results we conclude that the increase in the activity of glucokinase induced by a KCl‐rich medium is at least partly due to an increase in the concentration of Cl−, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.
Zonation of cytochrome P450 isozyme expression and induction in rat liver
Authors: BÜHLER Rolf , LINDROS Kai O. , NORDLING Åsa , JOHANSSON Inger , INGELMAN‐SUNDBERG Magnus 
Published: March 2005

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques. Immunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6–8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2E1 and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2. These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone‐restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenous‐restricted (CYP2B1/2, CYP2E1 and CYP3A1); perivenous‐dominated (CYP2A1) and panacinar (CYPEtOH2).Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the periportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial perivenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became periportal‐dominated. Acetone induced CYP2E1, CYP2C11 and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the periportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression.
Priming of human polymorphonuclear leukocytes with granulocyte‐macrophage colony‐stimulating factor involves protein kinase C rather than enhanced calcium mobilisation
Authors: SCHATZ‐MUNDING Monika , ULLRICH Volker 
Published: March 2005

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N‐formyl‐methionyl‐leucyl‐phenylalanine, fMLP) or a Ca2+‐ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5‐lipoxygenase.Preincubation of polymorphonuclear leukocytes with GM‐CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca2+‐threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350–400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells.Protein biosynthesis inhibitors like actinomycin D (10 γg/ml) and cycloheximide (50 γg/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM‐CSF after stimulation with ionomycin.The priming effect of GM‐CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultanous pretreat‐ment with PMA and GM‐CSF and stimulation with either fMLP or ionomycin.These results provide evidence that the enhanced arachidonic acid release in GM‐CSF‐primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.
Probing intercalation and conformational effects of the anticancer drug 2‐methyl‐9‐hydroxyellipticinium acetate in DNA fragments with circular dichroism
Authors: MONNOT Monique , MAUFFRET Olivier , LESCOT Elie , FERMANDJIAN Serge 
Published: March 2005

Circular dichroism was applied to the analysis of drug‐DNA associations. With the octanucleotide d(TGACGTCA) (octanucleotide I), which is the cAMP‐responsive element (CRE) in gene promoters and its reverse d(ACTGCAGT) (octanucleotide II), it was demonstrated that the anticancer polyaromatic agent celiptium intercalates into DNA base pairs with its long direction perpendicular to both the DNA‐helix axis and the base‐pair long axis and induces larger conformational changes in the CpG‐containing octanucleotide I CRE than in its reverse‐sequence octanucleotide II.It was concluded that CD is a powerful and sensitive technique to discriminate between drug‐binding modes of DNA, to define the geometry of the chromophore inserted into base pairs and, finally, to measure sequence‐dependent conformational changes induced by intercalation in DNA.We anticipate that these studies will contribute to a better understanding of the molecular bases that underlie the mechanism of action of those cytotoxic drugs which interfere with the DNA‐nuclear‐protein recognition.
Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10‐kb DNA fragment
Authors: VAN HUFFEL Christophe , DUBOIS Evelyne , MESSENGUY Francine 
Published: March 2005

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl‐phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327‐amino‐acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884‐amino‐acid polypeptide. The acetylglutamate kinase and acetylglutamate‐phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein.The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11− mutation. In contrast, both ornithine‐carbamoyltransferase‐encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization.The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.
Structural studies on fetal mucins from human amniotic fluid
Authors: HANISCH Franz‐Georg , PETER‐KATALINIC Jasna 
Published: March 2005

Mucins in human amniotic fluid are represented by two distinct molecular species, FM‐1 and FM‐2, with apparent molecular masses of 700 and 570 kDa, respectively, in SDS/polyacrylamide gradient gels. FM‐1 and FM‐2 were isolated by preparative SDS/PAGE to apparent homogeneity and subjected to structural studies on their carbohydrate portions. The carbohydrate compositions of the mucin species differed only marginally and exhibited significant amounts of mannose.O‐linked core‐region glycans on human amniotic mucin‐derived pronase‐stable glycopeptides were analyzed after reductive β‐elimination and purification on HPLC by a combination of methylation analysis, electron‐impact mass spectrometry of permethylated oligosaccharide alditols and fast‐atom‐bombardment mass spectrometry of acetylated or methylated alditols (positive‐ion mode) or alditol‐derived neoglycolipids (negative‐ion TLC‐MS). The primary structures of major monosaccharides to tetrasaccharides have been established which exhibit at their reducing termini core 1, core 2 and core 3 sequences, as follows.GalNAc‐ol  (N1)Galβ(1–3)GalNAc‐ol  (N3)Fucα(1–2)Galβ(1–3)GalNAc‐ol  (N4)Galβ(1–3)GlcNAcβ(1–3)GalNAc‐ol  (N5)Galβ(1–4)GlcNAcβ(1–3)GalNAc‐ol  (N5)Galβ(1–3)[GlcNAcβ(1–6)]GalNAc‐ol  (N6)Galβ(1–4)[Fucα(1–3)]GlcNAcβ(1–3)GalNAc‐ol  (N7)Galβ(1–3)[Galβ(1–4)GlcNAcβ(1–6)]GalNAc‐ol  (N9)
Glutamate mutase from Clostridium cochlearium
Authors: LEUTBECHER Ulrich , BÖCHER Reinhard , LINDER Dietmar , BUCKEL Wolfgang 
Published: March 2005

Both components, E and S, of the adenosylcobalamin‐(coenzyme B12)‐dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50‐kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260–3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide‐like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Coα‐[α‐(aden‐9‐yl)]‐cob(II)amide. A typical cob(II)amide signal at g= 2.23 with hyperfine and superhyperfine splitting was observed by EPR pectroscopy. A cobamide content of about 0.43 mol/mol 50‐kDa subunit was determined by cyanolysis.Substitution of the migrating hydrogen at C‐4 of glutamate by fluorine yielded the0potent competitive inhibitor (2S,4S)‐4‐fluoroglutamate (Ki= 70 μM). (2R,3RS)‐3‐Fluoroglutamate (Ki= 600 μM) was also inhibitory. The competitive inhibition by 2‐methyleneglutarate (Ki= 400 μM) and (S)‐3‐methylitaconate (Ki= 100 μM) but not by (RS)‐2‐methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N‐terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.
Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase
Authors: ROCHER Corinne , LALANNE Jean‐Louis , CHAUDIÈRE Jean 
Published: March 2005

We previously constructed plasmids for synthesis of glutathione‐peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active‐site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [CysIGPx and [SerIGPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [CysIGPx mutant exhibited a significant GPx activity, unlike the [SerIGPx mutant.Purification of [Cys]GPx was performed in two steps of ion‐exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel‐filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000‐fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa, of 8.3, presumably that of the active‐site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.
Calponin and SM 22 isoforms in avian and mammalian smooth muscle
Authors: GIMONA Mario , SPARROW Malcolm P. , STRASSER Peter , HERZOG Monika , SMALL J. Victor 
Published: March 2005

Calponin is a basic smooth‐muscle‐specific protein capable of binding to F‐actin, tropomyosin and calmodulin in vitro. Using two‐dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a PI identical to the most basic adult a variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees‐Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985–5991], which has sequence motifs related to calponin, displays a similar isoform pattern during development; one isoform (a) is present in the embryo and three in the adult.In living smooth‐muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32P04, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two‐dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65–68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10 148–10 1551]. In vitro translation of porcine and chicken smooth‐muscle mRNA produced only a single (α) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the α isoform and a second more basic spot, not observed in tissue samples.Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down‐regulated like calponin, metavinculin and heavy‐caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the α SM 22 isoform during smooth‐muscle phenotype modulation in vitro.
Structure of neuropeptide Y dimer in solution
Authors: COWLEY David J. , HOFLACK Jan M. , PELTON John T. , SAUDEK Vladimir 
Published: March 2005

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11 – 36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N‐terminus (residues 1–9) appears as an unstructured mobile segment.Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 ± 0.6 μM at pH 2–8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.
Tissue distribution of constitutive and induced soluble peroxidase in rat
Authors: DE Prabir K. 
Published: March 2005

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen‐induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane‐bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases.As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane‐bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5–3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane‐bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross‐react with lactoperoxidase it did interact with lactoperoxidase on Western blot.The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane‐bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue‐specific regulation of gene expression at the molecular level.