bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of an Intracellular Catalase‐Peroxidase from Penicillium Simplicissimum
Authors: Fraaije Marco W. , Roubroeks Hanno P. , Hagen Wilfred R. , Van Berkel Wiliem J. H. 
Published: August 2004

The first dimeric catalase‐peroxidase of eucaryotic origin, an intracellular hydroperoxidase from Penicillium simplicissimum which exhibited both catalase and peroxidase activities, has been isolated. The enzyme has an apparent molecular mass of about 170 kDa and is composed of two identical subunits. The purified protein has a pH optimum for catalase activity at 6.4 and for peroxidase at 5.4. Both activities are inhibited by cyanide and azide whereas 3‐amino‐1,2,4‐triazole has no effect. 3,3′‐Diaminobenzidine, 3,3′‐dimethoxybenzidine, guaiacol, 2,6‐dimethoxyphenol and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) all serve as substrates. The optical spectrum of the purified enzyme shows a Soret band at 407 nm. Reduction by dithionite results in the disappearance of the Soret band and formation of three absorption maxima at 440, 562 and 595 nm. The prosthetic group was identified as a protoheme IX and EPR spectroscopy revealed the presence of a histidine residue as proximal ligand.In addition to the catalase‐peroxid, an atypical catalase which is active over a broad pH range was also partially purified from P. simplicissimum. This catalase is located in the periplasm and contains a chlorin‐type heme as prosthetic group.
Isolation and Structures of Glycoprotein‐Derived Free Oligosaccharides from the Unfertilized Eggs of Scyliorhinus Caniculus
Authors: Plancke Yves , Delplace Florence , Wieruszeski Jean‐Michel , Maes Emmanuel , Strecker Gérard 
Published: August 2004

As previously reported [Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H. & Inoue, Y. (1989) J. Biol. Chem. 264, 1623–1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K. & Inoue, Y. (1989) J. Biol. Chem. 264, 18520–18526], the unfertilized eggs of two different species of fresh‐water fish, Plecoglossus altivelis and Tribodolon hakonensis, contain relatively large amounts of free sialooligosaccharides. These oligosaccharides were found to derive from glycophosphoproteins, owing to the activity of a peptide –N4‐(N ‐acetyl‐β‐d‐glucosaminyl)asparagine amidase [Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1992) J. Biol. Chem. 267, 24287–24296; Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1991) J. Biol. Chem. 266, 22110–22114].Here we describe a new type of free oligosaccharides, isolated from unfertilized eggs of Scyliorhinus caniculus. From the structural analysis, based upon 1H‐NMR spectroscopy, the following glycan units are proposed.
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Activation of Gene Transcription by Prostacyclin Analogues is Mediated by the Peroxisome‐Proliferators‐Activated Receptor (PPAR)
Authors: Hertz Rachel , Berman Ina , Keppler Dietrich , Bar‐Tana Jacob 
Published: August 2004

Xenobiotic amphipathic carboxylates, known collectively as hypolipidemic peroxisome proliferators (e.g., aryloxyalkanoic acids), or native long‐chain fatty acids induce liver peroxisome proliferation and other biological activities. This broad spectrum of effects results from modulation of transcription of specific genes mediated by binding of peroxisome‐proliferators‐activated receptors (PPAR) to respective sequence‐specific promoter elements (PPRE). The broad specificity and relatively low potency of reported hypolipidemic peroxisome proliferators prompted us to search for specific highly potent peroxisome proliferators. Here we report that stable prostacyclin analogues may act in such a manner.mPPARα‐mediated expression of a reporter gene linked to the peroxisomal rat acyl‐CoA oxidase promoter was dose‐dependently induced by carbaprostacyclin and iloprost. The ED50 for carbaprostacyclin was 25 nM, and carbaprostacyclin was therefore 25‐fold and 200‐fold more effective than the most potent xenobiotic (5,18,11,14‐eicosatetraynoic acid) and native (arachidonic acid) inducers, respectively. Induction was further increased by cotransfecting the cells with mPPARα and an expression vector for retinoic acid‐X‐receptor.PPAR‐mediated activation of gene expression by prostacyclin analogues was specific for PPAR and was not observed using other members of the superfamily.No activation of gene expression was induced by other prostaglandins or leukotrienes at concentrations 100‐fold higher than those of the prostacyclin analogues.Induction of gene expression by prostacyclin analogues was inhibited in cells transfected with the long‐chain‐acyl‐CoA synthase, indicating that the acidic form of prostacyclin, rather than the respective CoA derivative or a metabolite derived thereof, serves as the activator of the PPAR/PPRE transduction pathway.Hence, PPAR‐mediated modulation of gene transcription by prostacyclins may form the basis for their novel role as regulators of gene expression. Xenobiotic hypolipidemic peroxisome proliferators and native long‐chain fatty acids seem to exploit the PPAR/PPRE transduction pathway used by prostacyclin.
The Incorporation of Arachidonic Acid into Triacylglycerol in P388D1 Macrophage‐Like Cells
Authors: Balsinde Jesús , Dennis Edward A. 
Published: August 2004

When P388D1 cells are incubated in media containing 10 μM [3H] aragudibuc acid (4, Ach), the tabel is rapidly incorporated into phospholipids and triacylglycerol. However, incorporation of [3H] 4, Ach into phospholipids clearly precedes incorporation into triacylglycerol, indicating that the phospholipid pool constitutes the primary metabolic fate of the 4 Ach via a remodelling pathway. In contrast, [3H]4, Ach is incorporated into triacylglycerol almost exclusively via de novo synthesis, as evidenced by studies using propranolol, a phosphatidate phosphohydrolase inhibitor. This compound induced a time‐dependent and concentration‐dependent increase in the levels of [3H] 4 Ach‐containing phosphatidate that is directly correlated with a dccreasc in the levels or [3H] 4, Ach‐containing triacylglycerol. In addition, no change in the levels of [3H]4, Ach‐containing diacylglycerol and monoacylglycerol were apparent along the time course of fatty acid incorporation into triacylglycerol. However, a sharp and transient accumulation of cell‐associated free [3H]4 Ach wits detected shortly after exposure of the cells to the radioactive fatty acid.Collectively, the results reported herein suggest that free 4, Ach availability determines the patterns of incorporation and distribution of this fatty acid among the various lipid classes of P388D, cells.
The Gene, Protein and Glycan Structures of Laccase from Pleurotus ostreatus
Authors: Giardina Paola , Aurilia Vincenzo , Cannio Raffaele , Marzullo Liberato , Amoresano Angela , Siciliano Rosa , Pucci Piero , Sannia Giovanni 
Published: August 2004

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification.The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G. (1993) Appl. Microbiol. Biotechnol. 39, 632–636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast‐atom‐bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product.The protein was N‐glycosylated at Asn444. Glycan analysis showed the presence of only a high‐mannose structure containing varying numbers of mannose residues. The presence of O‐linked oligosaccharides as well as other post‐translational modification could be ruled out by the miss analysis.
Cyclic AMP and Fatty Acids Increase Carnitine Palmitoyltransferase I Gene Transcription in Cultured Fetal Rat Hepatocytes
Authors: Chatelain Florence , Kohl Claude , Esser Victoria , Mcgarry J. Denis , Girard Jean , Pegorier Jean‐Paul 
Published: August 2004

In the rat, the gene for liver mitochondrial carnitine palmitoyltransferase I (CPT I), though dormant prior to birth, is rapidly activated postnatally. We sought to elucidate which hormonal and/or nutritional factors might be responsible for this induction. In cultured hepatocytes from 20‐day‐old rat fetus, the concentration of CPT I mRNA, which initially was very low, increased dramatically in a dose‐dependent manner after exposure of the cells to dibutyryl cAMP (Bt2cAMP). Similar results were obtained when long‐chain fatty acids (LCFA), but not medium‐chain fatty acids, were added to the culture medium. The effects of Bt2cAMP and LCFA were antagonized by insulin, also dose dependently. In contrast, CPT II gene expression, which was already high in fetal hepatocytes, was unaffected by any of the above manipulations.Bt2cAMP stimulated CPT I gene expression even when endogenous triacylglycerol breakdown was suppressed by lysosomotropic agents suggesting that the actions of cAMP and LCFA were distinct. Moreover, half‐maximal concentrations of Bt2cAMP and linoleate produced an additive effect on CPT I mRNA accumulation. While linoleate and Bt2cAMP stimulated CPT I gene transcription by twofold and fourfold, respectively, the fatty acid also increased the half‐life of CPT I mRNA (50%).When hepatocytes were cultured in the presence of 2‐bromopulmitate, (which is readily converted by cells into its non‐metabolizable CoA ester) CPT I mRNA accumulation was higher than that observed with oleate or linoleate. Similarly, the CPT I inhibitor, tetradecylglycidate, which at a concentration of 20 μM did not itself influence the CPT I mRNA level, enhanced the stimulatory effect of linolcate. The implication is that induction of the CPT I message by LCFA does not require mitochondrial metabolism of these substrates; however, formation of their CoA esters is B necessary step.Unlike linoleate, the peroxisome proliferator, clofibrate, increased both CPT I and CPT II mRNA levels and neither effect was offset by insulin. It thus appears that the mechanism of action of LCFA differs from that utilized by clofibrate, which presumably works through the peroxisome proliferator activated receptor.We conclude that the rapid increase in hepatic CPT I mRNA level that accompanies the fetal to neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled with elevation of the liver content of CAMP.
Surface‐Associated Material from the Bacterium Actinobacillus actinomycetemcomitans Contains A Peptide Which, in Contrast to Lipopolysaccharide, Directly Stimulates Fibroblast Interleukin‐6 Gene Transcription
Authors: Reddi Krisanavane , Nair Sean P. , White Peter A. , Hodges Stephen , Tabona Peter , Meghji Sajeda , Poole Stephen , Wilson Michael , Henderson Brian 
Published: August 2004

The oral commensal Gram‐negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface‐associated material. This material contained potent bone‐resorbing activity. We now report that surface‐associated material is also a potent stimulator of cytokines, and in particular, interleukin‐6 (IL‐6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL‐6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL‐1, IL‐6 and tumour necrosis factor (TNF) α synthesis, surface‐associated material stimulated gingival fibroblasts to synthesize only IL‐6, with no induction of IL‐1 or TNF (the normal inducers of IL‐6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL‐1 or TNF in surface‐associated‐material‐stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS‐stimulated cells. Neutralizing antibodies to IL‐1 and/or TNF or the natural IL‐1 receptor antagonist (IL‐1ra) inhibited enteric LPS‐induced IL‐6 synthesis, but did not inhibit surface‐associated‐material‐induced synthesis. In addition, dexamethasone, which completely suppressed LPS‐induced IL‐6 synthesis, only inhibited surface‐associated‐material‐induced IL‐6 synthesis by 50%. This suggests that the active constituent in the surface‐associated material stimulates IL‐6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS.The IL‐6‐stimulating activity of the surface‐associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion‐exchange, reverse‐phase and size‐exclusion HPLC. The active moiety is a peptide of molecular mass 2 kDa which may be the product of a bacterial short open reading frame.
Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase
Authors: Marković‐Housley Zora , Schirmer Tilman , Hohenester Erhard , Khomutov Alex R. , Khomutov Radii M. , Karpeisky Marat Y. , Sandmeier Erika , Christen Philipp , Jansonius Johan N. 
Published: August 2004

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X‐ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5′‐phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single‐step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10−8 M to 10−6 M depending on the nature of the side‐chain group.The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of l‐aspartate in the open and closed enzyme conformation were determined at 0.23‐nm and 0.25‐nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the structures of both oxime complexes and the 2‐methylaspartate adduct. The crystal structures indicate that the interaction between the ω‐carboxylate of the inhibitor and Arg292* of the neighbouring subunit is mainly responsible for the attainment of near‐coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine‐derived inhibitors, even those containing a carboxylate group, do not induce the ‘domain closure’ in solution. This is probably due to the absence of the α‐carboxylate group in the monocarboxylic hydroxylamine‐derived inhibitors, emphasizing that both carboxylates of the substrates l‐Asp and l‐Glu are essential for stabilizing the closed form of aspartate aminotransferase.
Microheterogeneity of the Hydrophobic and Hydrophilic Part of the Glycosylphosphatidylinositol Anchor of Alkaline Phosphatase from Calf Intestine
Authors: Armesto Julio , Hannappel Ewald , Leopold Klaus , Fischer Werner , Bublitz Renate , Langer Lydia , Cumme Gerhard A. , Horn Anton 
Published: August 2004

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl‐(Etn‐P)2‐glycosyl‐PtdIns with HF generated 8 glycosyl‐Ins species the largest of which (G1 and G2) have the following proposed structures:
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G3 and G5 are lower homologues of G1 and G2, respectively, being one αl‐2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N ‐acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc‐Ins species are either substituted with an N ‐acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular‐mass determination by matrix‐assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse‐phase HPLC technique using fluorescent fluoren‐9‐yl‐methoxy‐carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level.We observed recently that native alkaline phosphatase separates on octyl‐Sepharose into four distinct fractions of increasing hydrophobicity (F1–F4). Here we show that all four fractions contain G1–G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl‐PtdIns–anchored proteins of mammalian origin.
Synthetic Alleles at Position 121 Define a Functional Domain of Human Interleukin‐1β
Authors: Ambrosetti Davide‐Carlo , Palla Emanuela , Mirtella Adriana , Galeotti Cesira , Solito Egle , Navarra Pierluigi , Parente Luca , Melli Marialuisa 
Published: August 2004

The non‐conservative substitution of the tyrosine residue at position 121 of human interleukin‐1β (IL‐1β generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T‐cells proliferation and (c) activation of interleukin‐6 (TL‐6) gene expression. It is generally accepted that these functions are mediated by the type‐I interleukin‐1 receptor (IL‐1RI). However, the mutant proteins maintain the binding affinity to the types‐I and II IL‐1 receptors, which is the same as the control IL‐1β, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally.Thus we have identified a new functional site of IL‐1β different from the known receptor binding region, responsible for fundamental IL‐1β functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short‐term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL‐1β molecule, possibly binding to more than one receptor chain, trigger different signals.
Phosphorylation of Plant Proteins and the Identification of Protein‐Tyrosine Kinase Activity in Maize Seedlings
Authors: Trojanek Joanna , Ek Pia , Scoble Judith , Muszyńska Grażyna , Engström Lorentz 
Published: August 2004

Phosphotyrosine was Found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins.Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark‐grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40–65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).
trans ‐Acting Factors, Detoxication Enzymes and Hepatitis B Virus Replication in a Novel Set of Human Hepatoma Cell Lines
Authors: Le Jossic Catherine , Glaise Denise , Corcos Laurent , Diot Christian , Dezier Jean‐François , Fautrel Alain , Guguen‐Guillouzo Christiane 
Published: August 2004

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver‐specific mRNAs and DNA‐binding activity of ubiquitous and liver‐enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3‐methylcholanthrene‐inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA‐binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF‐1 and HNF‐4‐like DNA‐binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1‐like DNA‐binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer‐binding protein and HNF‐3‐like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis‐B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis‐B virus replication and virion production, in agreement with the notion that HNF‐1 activity is necessary for viral replication.We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis‐B virus replication.
Developmental Expression of CYP2E1 in the Human Liver
Authors: Vieira Isabel , Sonnier Michelle , Cresteil Thierry 
Published: August 2004

Cytochromes P‐450 are responsible for the biotransformation of drugs and other hydrophobic molecules by the liver. Several isoforms coexist which display an asynchronous onset during the perinatal period suggesting the involvement of multiple mechanisms of regulation. In this paper, we have shown that the CYP2E1 protein and its associated activity could not be detected in the fetal liver and rise during the first few hours following birth independently of the gestational age (between 25–40 weeks). During this period, the CYP2E1 RNA content remains fairly low: the stabilization of the low amount of existing CYP2E1 protein by endogenous ketone bodies could explain the early neonatal rise of the protein level. From 1 month to 1 year, the protein content gradually increases and is accompanied by the accumulation of CYP2E1 RNA, suggesting a transcriptional activation of the gene during the late neonatal period.We examined the methylation status of CpG residues in the 5′ flanking region, first exon and first intron of CYP2E1 gene cleaved with Hpa II/Msp I. Genomic DNA from fetal liver shows several hypermethylated spots in the first‐exon–first‐intron region, which progressively disappear in neonatal samples. We conclude that during the neonatal period, the accumulation of hepatic CYP2E1 RNA is correlated with the degree of methylation at the 5′ end of the CYP2E1 gene.