bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Catalytic Properties of Two Manganese Peroxidase Isoenzymes from Pleurotus eryngii
Authors: Martínez María J. , Ruiz‐Dueñas Francisco J. , Guillén Francisco , Martínez Ángel T.
Published: August 2004

The ligninolytic basidiomycetes Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor‐caju did not exhibit detectable levels of manganese peroxidase (MP) when grown in liquid media with ammonium tartrate as N source. However, after examination of cells grown on different organic N‐based media, high MP activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of P. eryngii. Moreover, Mn2+ supplementation was not used to produce MP, since all Mn2+ concentrations assayed (1–4000 μM) inhibited production of this enzyme in liquid medium.

Two MP isoenzymes were purified to homogeneity from shaken or stationary cultures of P. eryngii grown in peptone medium. The purification process (which included chromatography on Biorad Q‐cartridge, Sephacryl S‐200 and Mono‐Q) attained 56% activity yield with a purification factor of 25. The isoenzymes differed in pI (3.75 and 3.65), N‐terminal sequence and some catalytic properties. They were in some aspects (e.g., molecular mass of 43 kDa) similar to Phanerochaete chrysosporium MP but exhibited some distinct characteristics, including Mn2+ ‐independent peroxidase activities against 2,6‐dime‐thoxyphenol and veratryl alcohol, and higher resistance to H2O2. Recent studies have shown that MP are ubiquitous enzymes in ligninolytic fungi, but the results obtained suggest that differences in catalytic properties probably exist between different Mn2+ ‐oxidizing peroxidases produced by these fungi.
Characterization of the Core Promoter of the Na+/K+‐ATPase α1 Subunit Gene
Authors: Kawakami Kiyoshi , Masuda Kazuyuki , Nagano Kei , Ohkuma Yoshiaki , Roeder Robert G.
Published: August 2004

We have analyzed the core promoter element of the Na+/K+‐ATPase α1 subunit gene by means of an in vitro transcription system composed of a HeLa nuclear extract. 5′‐deletion and 3′‐deletion analyses revealed that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gene (−102 to −61), and that the 3′ boundary of the minimal promoter element does not extend beyond +5. Analysis of linker‐substitution mutations and point mutations revealed that the TATA‐like sequence (−33 to −26) is required for upstream‐sequence‐dependent transcription whereas linker‐substitution mutations and point mutations near +1 did not abolish transcription.

The gene was found to be transcribed by RNA polymerase III when phosphocellulose column fractions were assayed. Deletion analysis mapped the minimal RNA‐polymerase‐III–specific promoter element from −49 to +17. The phosphocellulose 0.3‐M‐KCl fraction is absolutely required for transcription by RNA polymerase III, while the 0.85‐M‐KCl fraction represses aberrant transcription from incorrect initiation sites. Analysis of linker‐substitution mutations indicated that the TATA‐like sequence is required for RNA‐polymerase‐III–specific transcription. Although point mutations in the 5′ half of the TATA‐like sequence did not affect transcription, those in the 3′ half shifted the transcription initiation site 3 bp upstream. The results suggest that the Na+/K+‐ATPase α1 subunit gene promoter contains a TATA‐like sequence which can direct transcription by RNA polymerase III in vitro. The mechanism of alternative regulation of RNA polymerase II and RNA polymerase III is discussed.
Synthesis and Differential Properties of Creatine Analogues as Inhibitors for Human Creatine Kinase Isoenzymes
Authors: Min Kyung‐Lyum , Steghens Jean‐Paul , Henry Robert , Doutheau Alain , Collombel Christian
Published: August 2004

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed‐type inhibition similar to that of creatine. Among these compounds 2,6‐dichlorobenzylguanidine (Ki= 5.6 mM and 39.8 mM for muscle‐type and brain‐type creatine kinases, respectively) and 3–(2,6‐dichlorophenyl)propylguanidine (Ki, = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle‐ and brain‐type creatine kinases.

Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine‐binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.
Cyclin‐Dependent Kinase‐2 (Cdk2) Forms an Inactive Complex with Cyclin D1 Since Cdk2 Associated with Cyclin D1 is Not Phosphorylated by Cdk7–Cyclin‐H
Authors: Higashi Hideaki , Suzuki‐Takahashi Ikuko , Saitoh Shio , Segawa Kaoru , Taya Yoichi , Okuyama Akira , Nishimura Susumu , Kitagawa Masatoshi
Published: August 2004

Cyclin‐dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor‐suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2–cyclin‐D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2–cyclin‐D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk–cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2–cyclin‐D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F‐1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2–cyclin‐E and Cdk2–cyclin‐A phosphorylated these proteins. Moreover, the Cdk2–cyclin‐D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2–cyclin‐D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under‐shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2–cyclin‐D1 complex in the baculovirus system. In human WI‐38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid‐G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7–cyclin‐H, while Cdk2 bound to cyclin E, as well as free Cdk2, was phosphorylated by Cdk7–cyclin‐H. Additionally, Cdk2 phosphorylated by Cdk7–cyclin‐H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7–cyclin‐H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.
Stereochemical Requirements of Chitin Synthase for Ligand Binding at the Allosteric Site for N‐Acetylglucosamine
Authors: Horsch Markus , Mayer Christoph , Rast Dora M.
Published: August 2004

The substrate kinetics of chitin synthase (CS) were non‐Michaelian, irrespective of the type of enzyme preparation studied (105‐S chitosomal CS, and 16‐S CS ex walls), even in the presence of saturating GlcNAc. An unexplained idiosyncrasy of this enzyme, which is likely to be responsible for this phenomenon, is evident from the striking non‐linearity of product deposition with time at low substrate or low enzyme concentrations, particularly in the absence of GlcNAc. The possibility can be excluded that this non‐linearity is due to the formation of soluble by‐products or intermediates in the form of chito‐oligomers, as shown by HPLC/pulsed amperometric detection analysis. Additional evidence was sought for the tenet that CS is homotropically‐heterotropically regulated, at least under steady‐state reaction conditions. Substrate kinetic curves established from rate data for the linear reaction phase only were used for modelling. These could be reasonably well parameterised on the basis of the Monod mathematical model for co‐operative ligand binding. Within a series of test compounds used to assess the stereochemical conditions of the allosteric site of CS for effector binding, N‐acetylglucosaminono‐1,5‐lactone oxime excelled. Requirements for effector binding are as follows: (a) an aminoglucopyranose skeleton with the amino function acetylated, and (b) a single‐bonded oxo‐function present at C(1), which is preferentially a hydrogen bond donor, that may be equatorially spaced off, but must not be α‐anomeric. The implications of these findings for chitin synthesis in vivo are discussed in terms of a mechanistically based fitness of CS to operate efficiently under vastly different combinations of substrate and effector concentrations as well as with respect to the conditions under which chitin synthase could be coordinately linked to the catabolism of chitin.
Colocalization of the Dihydropyridine Receptor, the Plasma‐Membrane Calcium ATPase Isoform 1 and the Sodium/Calcium Exchanger to the Junctional‐Membrane Domain of Transverse Tubules of Rabbit Skeletal Muscle
Authors: Sacchetto Roberta , Margreth Alfredo , Pelosi Michele , Carafoli Ernesto
Published: August 2004

The subcellular distribution of the calmodulin‐stimulated plasma‐membrane Ca2+‐ATPase (PMCA) has been studied in rat and rabbit skeletal muscle cells by indirect (calmodulin gel overlays) and direct (Western blotting with specific antibodies) methods. It has also been studied in situ in immunocytochemistry experiments. The distribution of PMCA has been compared with that of the Na+/Ca2+ exchanger and of the dihydropyridine receptor, which has been studied by Western blotting with specific antibodies. Both PMCA and the Na+/Ca2+ exchanger had a dual localization, i.e., they were found in the plasma membrane and in the transverse‐tubule fractions of the two main types of skeletal muscles studied. The pump and the exchanger were not diffusely distributed in the transverse‐tubule‐membrane system, but specifically confined to the membrane domain where the dihydropyridine receptor was also localized, i.e., the junctional membrane. Experiments with isoform‐specific antibodies have shown that the pump isoform expressed in skeletal muscle is PMCA 1.
Decreased Rates of Replicon Initiation in Mammalian Cells
Authors: Tsvetkov Lyuben , Russev George
Published: August 2004

We have designed an assay to measure the rate of initiation of DNA synthesis in Friend erythroleukemia cells and have shown that this parameter is reduced by y‐radiation and treatment with 4′‐demethyl‐epipodophyllotoxin‐9‐(4,6‐O‐ethylene‐β‐D‐glucopyranoside) (VP‐16). It is concluded, that double‐strand breaks in DNA are the immediate cause for this effect. The decrease in the rate of replicon initiation is affected differently by different agents such as cis‐diamminedichloroplatinum(II), cycloheximide, staurosporine, and 3‐aminobenzamide. The analysis of these results indicates that the observed partial decrease of the rate of DNA initiation is most probably transmitted from the site of damage to the initiation site by one or more phosphorylation/dephosphorylation steps. It does not require de novo synthesis of protein factors, but is probably dependent on poly(ADP‐ribosyl)ation of chromatin at the site of DNA breaks.
Biotin‐Labelled and Photoactivatable Aldosterone and Progesterone Derivatives as Ligands for Affinity Chromatography, Fluorescence Immunoassays and Photoaffinity Labelling
Authors: Eisen Christoph , Meyer Christiane , Dressendörfer Regina , Strasburger Christian , Decker Heinz , Wehling Martin
Published: August 2004

New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3‐(O ‐carboxymethyl)‐oximino‐(3‐[125I]iodo‐4‐azidosalicylamidobutylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid.

Aldosterone was labelled with biotin and used as a tracer in a time‐resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibody‐binding site, with almost no detectable cross‐reactivity for other steroids. Biotin‐labelled progesterone was immobilized by avidin‐agarose and used for affinity chromatography. This yielded a more than 20‐fold enrichment of an anti‐progesterone polyclonal antibody.

These results demonstrate that derivatives of steroids are particularly useful for the development of nonradioactive assays for the determination of natural steroids and may be also useful for the detection of specific binding sites in biological material such as plasma membranes.
Synthesis, Characterization and Preliminary Crystallographic Data of N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino‐acid Oxidase from Pig Kidney, a Semi‐Synthetic Oxidase
Authors: Stocker Achim , Hecht Hans‐Jürgen , Bückmann Andreas F.
Published: August 2004

The FAD analogue, N6‐(6‐carboxyhexyl)‐FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi‐synthetic oxidase, N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase, was prepared by reacting the succinimido ester of N6‐(6‐carboxyhexyl)‐FAD with apo‐d‐amino‐acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi‐synthetic and fully active N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)‐MS and peptide mapping.

Presumably, the N6‐(6‐carbamoylhexyl)‐FAD moiety of this semi‐synthetic d‐amino‐acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non‐covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi‐synthetic DAAO, Vmax(app) = 17.7 μmol min‐1 mg‐1; KM(app) = 4.5 mM; native holo‐DAAO, Vmax= 12.2 μmol min‐1 mg‐1; KM= 1.8 mM). Compared with the native holo‐d‐amino acid oxidase, this new semi‐synthetic N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase is a considerably more stable enzyme that shows meso‐thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6‐(6‐carbamoylhexyl)‐FAD‐d‐amino acid oxidase. The single crystals, obtained by the sitting‐drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a= 16.3 nm, c= 13.6 nm. The crystals diffract to 0.3‐nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two‐subunit quarternary structure of this semi‐synthetic D‐amino‐acid oxidase.
Acetylcholinesterase in Dendrobaena veneta (Oligochaeta: Opisthopora) is Present with Forms Sensitive and Insensitive to Phosphatidylinositol Phospholipase C
Authors: Talesa Vincenzo , Romani Rita , Rosi Gabriella , Giovannini Elvio
Published: August 2004

Three distinct acetylcholinesterases were detected in the annelid oligochaete Dendrobaena veneta. Two enzymes (α, β), copurified from a Triton‐X‐100‐soluble extract of whole animals by affinity (edrophonium‐Sepharose) chromatography, were separately eluted from a Sephadex G‐200 column. Gel‐filtration chromatography, sedimentation analysis and SDS/PAGE showed the α and β forms to be a globular dimer (110 kDa, 7.0 S) and a hydrophilic monomer (58 kDa, 5.0 S) respectively, both weakly linked to the cell membrane. The third form (γ), also purified to homogeneity by slower filtration through an edrophonium‐Sepharose matrix, proved to be an amphiphilic globular dimer (133 kDa, 7.0 S) with a phosphatidylinositol anchor giving cell membrane insertion, detergent (Triton X‐100, Brij 96) interaction and self‐aggregation. The α acetylcholinesterase showed a fairly low substrate specificity: the β form hydrolyzed propionylthiocholine at the highest rate and was inactive on butyrylthiocholine; the γ acetylcholinesterase, showing a marked active‐site specificity with differently sized substrates, was likely functional in cholinergic synapses. Studies with inhibitors showed incomplete inhibition of all three acetylcholinesterase by 1 mM eserine and different sensitivity for edrophonium or procainamide. The α and β forms, sensitive to 1,5‐bis(4‐allyldimethylammoniumphenyl)‐pentan‐3‐one dibromide, were unaffected by tetra(monoisopropyl)‐pyrophosphortetramide, while both these agents inhibited the γ enzyme. All three forms showed excess‐substrate inhibition by acetylthiocholine. Enzyme activity was histochemically localized in the nerve ring and its minor branches. Monomeric acetylcholinesterase (β) is likely the only form present in the ganglionic glial framework.
Construction and Functional Characterization of Recombinant Fusion Proteins of Human Lipoprotein Lipase and Apolipoprotein CII
Authors: Hoffmann Michael M. , Stoffel Wilhelm
Published: August 2004

The hydrolysis of triacylglycerols of chylomicrons and very low density lipoproteins by lipoprotein lipase (LPL) requires the presence of apolipoprotein (apo) CII as a cofactor. To obtain further information on the interaction of apo CII and LPL, we generated two fusion proteins consisting of the complete LPL molecule and the mature form of apo CII. The cDNAs of both proteins were either connected directly or by a segment encoding a 16‐amino‐acid linker peptide. The fused cDNAs were stably expressed in human embryonic kidney (HEK) 293 cells and the enzymic properties of the recombinant proteins were examined. The fusion proteins hydrolysed both emulsified long‐chain (lipase) triacylglycerol substrate and a water‐soluble short‐chain (esterase) fatty acid ester substrate (p ‐nitrophenylbutyrate), regardless of whether or not they contained the linker peptide. In the absence of exogenous apo CII, the fusion proteins had up to 3.5‐times higher basal activity than wild‐type LPL. Similar to wild‐type LPL, the fusion proteins were inhibited by 1 M NaCl, however less than wild‐type LPL. A polyclonal antibody specific for apo CII impaired their ability to hydrolyse triacylglycerol emulsions. A similar effect was seen when the tetrapeptide KGEE was used as inhibitor, which corresponds to the carboxy‐terminal four amino acids of apo CII.
Acetyl Xylan Esterase from Trichoderma reesei Contains an Active‐Site Serine Residue and a Cellulose‐Binding Domain
Authors: Margolles‐Clark Emilio , Tenkanen Maija , Söderlund Hans , Penttilä Merja
Published: August 2004

The axe 1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe 1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe 1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe 1 product was inactivated with phenylmethylsulfonyl fluoride. At its C‐terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.
Interferon‐Modulated Expression of Genes Encoding the Nuclear‐Dot‐Associated Proteins Sp100 and Promyelocytic Leukemia Protein (PML)
Authors: Grötzinger Thilo , Sternsdorf Thomas , Jensen Kirsten , Will Hans
Published: August 2004

Promyelocytic leukemia protein (PML) and Sp100 are transcription‐regulatory proteins which colocalize in discrete nuclear dots and play a role in autoimmunity, oncogenesis and virus‐host interaction. Interferons (IFNs) were shown previously to increase strongly the levels of Sp100 mRNA and protein. Here, we examined which mechanisms lead to upregulation of Sp100 gene expression and whether IFNs also increase expression of the promyelocytic leukemia (PML) gene. We found that both mRNA and protein levels of PML are also strongly upregulated by IFNs. In addition, new Sp100 and PML proteins were detected immunologically after IFN treatment of cells. Nuclear run‐on analysis revealed protein‐synthesis‐independent, rapid IFN‐enhanced transcription rates as well as synergistic activation of the Sp100 and PML genes by type‐I and type‐II IFNs. These data demonstrate that PML and Sp100 belong to the growing family of IFN‐stimulated genes (ISGs) upregulated most likely by the transcription factor ISGF3, and indicate that IFNs also qualitatively alter the expression of these two genes.
Adenosine Stimulates Calcium Influx in Isolated Rat Hepatocytes
Authors: Tinton Sandrine A. , Chow Sek C. , Buc‐Calderon Pedro M. , Kass George E. N.
Published: August 2004

The mechanism of stimulation of Ca2+ entry into hepatocytes by adenosine was investigated. When Fura‐2‐loaded hepatocytes were suspended in a nominally Ca2+‐free buffer, adenosine produced only a small transient increase in the cytosolic free Ca2+ concentration ([Ca2+Ji). However, on restoration of an extracellular Ca2+ concentration of 1.3 mM, a rapid increase in ([Ca2+]i) occurred, which indicates activation of a Ca2+‐influx pathway. Adenosine augmented the rate of Ca2+ influx triggered by maximally effective concentrations of thapsigargin or cAMP, but was without effect on the rate of Ca2+ entry that resulted from phospholipase‐C–linked‐receptor activation by maximally effective concentrations of vasopressin or ATP. However, in contrast to vasopression and ATP, adenosine did not stimulate Mn2+ entry. The rate of Mn2+ influx after stimulation of the hepatocytes with vasopressin was not increased by adenosine treatment. The stimulation of hepatocytes with adenosine did not result in significant accumulation of inositol phosphates or cAMP. Furthermore, the rate of adenosine‐induced Ca2+ entry in hepatocytes was only slightly reduced in the presence of the P1 purinoceptor antagonist 8‐phenyltheophylline. In contrast, the receptor‐mediated‐Ca2+‐entry antagonist SK&F 96365 nearly completely blocked the Ca2+‐entry response without any effect on internal‐Ca2+‐pool mobilisation by adenosine. It is concluded that adenosine activates the internal‐pool‐regulated pathway of Ca2+ entry and an additional pathway that appears comparable to the previously reported receptor‐dependent pathway, except that Mn2+ entry is not stimulated.
Molecular Cloning, Expression and Characterization of Mouse Leukotriene C4 Synthase
Authors: Lam Bing K. , Penrose John F. , Rokach Joshua , Xu Kongyi , Baldasaro Mathew H. , Austen K. Frank
Published: August 2004

Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A4 to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C4 synthase shares substantial amino acid identity in its consensus N‐terminal two‐thirds with 5‐lipoxygenase‐activating protein and has a region (residues 375–58) that exhibits 46% amino acid identity with a domain of this protein (residues 41–62) to which an inhibitor binds. We have now cloned mouse leukotriene C4 synthase cDNA using the polymerase chain reaction to screen a mouse pcDNA3 expression library with oligonucleotide primers based on the translated human leukotriene C4 synthase cDNA sequence. Mouse leukotriene C4 synthase cDNA is 667 bp in length, including the poly(A)‐rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150‐amino‐acid sequence of mouse leukotriene C4 synthase differs from the human enzyme by only 18 amino acids, of which 9 reside at the C terminus. The potential N‐glycosylation site, two protein kinase C phosphorylation sites, the two cysteine residues, and the putative inhibitor‐binding domain (substitutions Thr41→Ser and Tyr50→Phe) were conserved in mouse leukotriene C4 synthase. Northern blot analysis indicated that the leukotriene C4 synthase RNA transcript is widely distributed. The Km values for leukotriene A4 methyl ester, leukotriene A4 free acid and GSH were 7.6 μM, 3.6 μM and 1.6 mM, respectively, for purified human recombinant enzyme, and 10.3 μM, 2.5 μM and 1.9 mM, respectively, for purified recombinant mouse enzyme; the corresponding Vmax values were 2.5, 1.3 and 2.7 μmol−1· min−1· mg−1 protein, respectively, for human enzyme, and 2.3, 1.2 and 2.2 μmol · min−1· mg−1 protein, respectively, for mouse enzyme. The 5‐lipoxygenase‐activating‐protein inhibitor, MK‐886, was active against both human and mouse recombinant leukotriene C4 synthase with IC50 values of 3.1 μM and 2.7 μM, respectively. These findings confirm that the leukotriene C4 synthases belong to a gene family that includes the 5‐lipoxygenase‐activating protein and suggest that the C‐terminal domain of leukotriene C4 synthase may not be critical for its conjugation function.
Induction of CYP1A1 Gene by Benzimidazole Derivatives During Caco‐2 Cell Differentiation
Authors: Daujat Martine , Charrasse Sophie , Fabre Isabelle , Lesca Pierre , Jounaidi Youssef , Larroque Christian , Poellinger Lorenz , Maurel Patrick
Published: August 2004

The Caco‐2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer β‐naphthoflavone in the Caco‐2 cells at various culture stages. In addition, we characterized the Caco‐2 aryl‐hydrocarbon receptor. The protein‐synthesis inhibitor cyclo‐heximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either β‐naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco‐2 cells in long‐term cultures, we observed a difference in behavior between the classical inducer β‐naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to β‐naphthoflavone was long lasting. The Caco‐2 aryl‐hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco‐2 cells occurred with no direct binding of benzimidazole derivatives to the aryl‐hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic‐responsive element enhancer, with which the activated aryl‐hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl‐hydrocarbon receptor in the nuclei of the Caco‐2 cells exposed to these molecules was clearly demonstrated by gel‐retardation experiments. These results question about the mechanism of ligand‐independent activation of the aryl‐hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.
A Recombinant Insect‐Specific α‐Toxin of Buthus occitanus tunetanus Scorpion Confers Protection Against Homologous Mammal Toxins
Authors: Bouhaouala‐Zahar Balkiss , Ducancel Frédérique , Zenouaki Ilham , Ben Khalifa Rym , Borchani Lamia , Pelhate Marcel , Boulain Jean‐Claude , El Ayeb Mohamed , Ménez André , Karoui Habib
Published: August 2004

We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an α‐toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig‐binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant α‐toxin/I bacterial culture. The toxin, called Bot XIV, displays no toxicity towards mammals but is active towards insects as shown by its paralytic activity against Blatella germanica cockroach and by electrophysiological studies on Periplaneta americana cockroaches. The Bot XIV protein fused to two Z domains is highly immunogenic in mice and induces production of antisera that specifically recognize and neutralize highly toxic components that had been injected into mice. This fusion protein could be very useful for development of potent protective antisera against scorpion venoms.
Molecular Cloning of Endothelial, Inducible Nitric Oxide Synthase Gene from Rat Aortic Endothelial Cell
Authors: Iwashina Masatora , Hirata Yukio , Imai Taihei , Sato Kyoko , Marumo Fumiaki
Published: August 2004

We have isolated and sequenced clones of an inducible nitric oxide synthase (iNOS) from cDNA library of interleukin‐1β‐treated rat aortic endothelial cells (EC) completely free from other cell types. The cloned cDNA contains an ORF consisting of 3441 bp, which encodes 1147 amino acid residues. The deduced amino acid sequence contains putative binding sites for NADPH, FMN, FAD, calmodulin and heme. By comparison with amino acid sequences of other isoforms, rat EC iNOS is very similar (92% similarity) to mouse macrophage iNOS. There are four AUUUA motifs, potentially responsible for the instability of the mRNA, in 3′ non‐coding region of rat EC iNOS cDNA. Transient transfection of cultured rat vascular smooth‐muscle cells with a full‐length rat EC iNOS cDNA/SRα296 construct by electroporation resulted in massive NO production in proportion to the doses of cDNA used. Northern blot analysis using rat EC iNOS cDNA as a probe revealed that cycloheximide treatment led to a marked accumulation of iNOS mRNA in the presence and absence of interleukin‐1β. No appreciable decay in the cyclo‐heximide‐induced iNOS mRNA accumulation was observed, suggesting that blockade of de novo protein synthesis stabilizes mRNA. These results demonstrate that rat EC iNOS is identical (or very similar) to macrophage iNOS, and suggest that the EC iNOS gene is also regulated at the post‐transcriptional level.
The guanine‐nucleotide‐exchange Complex (EF‐1βγδ) of Elongation Factor‐1 Contains two Similar Leucine‐Zipper Proteins EF‐1δ, p34 Encoded by EF‐1δ1 and p36 Encoded by EF‐1δ2
Authors: Minella Odile , Mulner‐Lorillon Odile , Poulhe Robert , Bellé Robert , Cormier Patrick
Published: August 2004

We have cloned and sequenced a Xenopus cDNA referred to as EF‐1δ2. The cDNA is homologous to EF‐1δ1 encoding for EF‐1δ a protein of the guanine‐nucleotide exchange complex of elongation factor‐1 (EF‐1). The protein sequence deduced from the cDNA, contains the two characteristic features of EF‐1δ protein, the leucine‐zipper domain and the guanine‐nucleotide exchange domain. In vitro and in vivo translation leads to the production of a 36‐kDa protein from EF‐1δ2 and a 34‐kDa protein from EF‐1δ1. The clone EF‐1δ2 therefore encodes for authentic p36 protein of EF‐1βγδ complex, while EF‐1δ1 encodes for a newly characterised p34 protein of the leucine zipper family. Both EF‐1δ proteins are simultaneously present in oocytes extracts, at a molecular ratio around 1:10 for p34 versus p36 proteins. Both are associated in a macromolecular structure that is greater than 750 kDa upon gel filtration. The two proteins are targets for Cdc2 kinase in meiotic maturation.
Evidence for Cu(I) Clusters and Zn(II) Clusters in Neuronal Growth‐Inhibitory Factor Isolated from Bovine Brain
Authors: Bogumil Ralf , Faller Peter , Pountney Dean L. , Vašák Milan
Published: August 2004

Neuronal growth‐inhibitory factor (GIF), a central‐nervous‐system‐specific metallothionein‐like protein, has been isolated by means of an improved isolation procedure from bovine brain. The native protein contains 4–5 Cu+and 2–2.5 Zn2+, which results in an overall stoichiometry of 6–7 mol metal ions/mol protein. Native Cu, Zn‐GIF and the Zn2+‐substituted and Cd2+‐substituted metalloforms have been characterized by means of electronic‐absorption, CD, magnetic‐circular‐dichroism (MCD) and low‐temperature (77 K) Cu(I)‐luminescence spectroscopy. Analysis of the metal‐induced‐charge‐transfer transitions below 300 nm in the electronic‐absorption and CD spectra of Cu, Zn‐GIF revealed spectral features characteristic of metal‐thiolate coordination. The presence of formally spin‐forbidden 3d → 4s Cu(I)‐cluster‐centered transitions above 300 nm in the corresponding CD and MCD spectra indicate the existence of a Cu(l) cluster. The 77‐K luminescence spectrum of Cu, Zn‐GIF revealed two emissive bands at approximately 420 nm and 570 nm, which were reported also for Cu4clusters in mammalian Cu8‐‐metallothionein. By analogy with Cu8‐metallothionein, we propose the presence of a Cu4cluster with similar electronic structure in native GIF. However, the determined Cys/Cu1ratio of approximately 2:1 in Cu, Zn‐GIF is higher than the ratio found in mammalian Cu(I)‐metallothionein forms (approximately 1.6:1), which implies that the coordination geometry of Cu+‐binding sites is different in the Cu4cluster. The spectroscopic characterization of Zn2+‐substituted and Cd2+‐substituted GIF (6–7 metal ions/protein) showed CD and MCD features at positions identical to those reported for the well‐characterized mammalian Zn7‐metallothionein and Cd7‐metallothionein. Therefore, it is inferred that the cluster organization in GIF with divalent metal ions is comparable to that found in mammalian metallothioneins. The effect of metal ions on the protein structure with regard to the biological function of GIF is discussed.
Signaling Pathway Associated with Stimulation of CB2 Peripheral Cannabinoid Receptor
Authors: Bouaboula Monsif , Poinot‐Chazel Caroline , Marchand Jean , Canat Xavier , Bourrié Bernard , Rinaldi‐Carmona Murielle , Calandra Bernard , Le Fur Gérard , Casellas Pierre
Published: August 2004

Cannabinoids, known for their psychoactive effects, also possess immunomodulatory properties. The recent isolation and cloning of the G‐protein‐coupled peripheral cannabinoid receptor (CB2), mainly expressed in immune tissues, have provided molecular tools to determine how cannabinoid compounds may mediate immunomodulation. We here investigated the CB2 signaling properties using stably transfected Chinese hamster ovary cells expressing human CB2. First, we showed that stimulation by a cannabinoid agonist activated mitogen‐activated protein (MAP) kinase in time‐ and dose‐dependent manners. The rank order of potency for MAP kinase activation of cannabinoid agonists correlated well with their binding capacities. Second, we demonstrated that, following MAP kinase activation, cannabinoids induced the expression of the growth‐related gene Krox‐24, also known as NGFI‐A, zifl 268, and egr ‐1. Pertussis toxin completely prevented both MAP kinase activation and Krox‐24 induction, even more these responses appeared to be dependent of specific proteine kinase C isoforms and independent of inhibition of adenylyl cyclase. A similar coupling of CB2 to a mitogenic pathway and to the regulation of Krox‐24 expression was also observed in human promyelocytic cells HL60. Taken together, these findings provide evidence for a functional role of the CB2 receptor in gene induction mediated by the MAP kinase network.
Involvement of Src‐homology‐2‐domain‐containing Protein‐tyrosine Phosphatase 2 in T Cell Activation
Authors: Tailor Pankaj , Jascur Thomas , Williams Scott , Von Willebrand Maria , Couture Clément , Mustelin Tomas
Published: August 2004

Activation of resting T lymphocytes by ligands to the complex of T cell antigen receptor (TCR) and CD3 is initiated by a series of critical tyrosine phosphorylation and dephosphorylation events. Protein‐tyrosine kinases of the Syk, Src and Csk families and the CD45 protein‐tyrosine phosphatase (PTPase) are known to be involved in these early biochemical reactions. We have found that one of the two T‐cell‐expressed SH2‐domain‐containing PTPases, SHPTP2, is rapidly phosphorylated on tyrosine upon addition of anti‐CD3 mAbs. This response was absent in cells lacking the Src family kinase Lck. Concomitantly with tyrosine phosphorylation, SHPTP2 co‐immunoprecipitated with two unphosphorylated cellular proteins: phosphatidylinositol 3‐kinase p85 and Grb2. Binding of SHPTP2 to Grb2 occurred through the SH2 domain of Grb2, while the association between SHPTP2 and p85 seemed to be mediated through Grb2 as an intermediate. In addition, many other molecules associate with Grb2 and may thereby become juxtaposed to SHPTP2. Our results indicate that SHPTP2 participates actively at an early stage in TCR signaling and that its phosphorylation on tyrosine may direct a Grb2‐dependent association with selected substrates.
Transcription of Four Satellite DNA Subfamilies in Diprion pini (Hymenoptera, Symphyta, Diprionidae)
Authors: Rouleux‐Bonnin Florence , Renault Sylvaine , Bigot Yves , Periquet Georges
Published: August 2004

Four satellite DNA subfamilies Ps, Pv, Pv65 and Ec, resulting from the evolution of a common ancestral motif, were isolated and characterized in the genomic DNA of Diprion pini, a phytophagous of Pinus sylvestris. Consensus sequences were 148–312 bp long. Sequence analyses revealed that these satellite subfamilies have evolved from a 45‐bp ancestral motif. The amounts of each satellite in the genome (0–10%) and the accessibility of the DNA to restriction enzymes were sex dependent. The migration of each monomer in polyacrylamide gels and the electrophoretic migration of d(AT)n≥3 residues showed that all four satellite subfamilies are curved. Their transcription was analyzed using reverse transcription and the polymerase chain reaction. Three satellite DNA subfamilies were transcribed on both strands, and in both sexes. However, the female satellite DNAs seem to be more actively transcribed than those of males, indicating that transcription is not constitutive. The lack of any significant open reading frame in satellite monomers indicates that the RNA may function as structural or catalytic RNA rather than encoding protein.
Purification and Structural Characterization of Bovine Cathelicidins, Precursors of Antimicrobial Peptides
Authors: Storici Paola , Tossi Alessandro , Lenarčič Brigita , Romeo Domenico
Published: August 2004

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N‐terminal region while the C‐terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP‐28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES‐MS data from the purified proteins with their cDNA‐deduced sequences has revealed several common features of their primary sequence, such as the presence of N‐terminal 5‐oxoproline (pyroglutamate) residues and two disulfide bridges in a 1–2, 3–4 arrangement. The N‐terminal domains of the cathelicidins present one or two Asp‐Pro bonds, which are particularly acid‐labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine‐containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C‐terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence‐based secondary structure predictions and CD results indicate for cathelicidins a 30–50% content of extended conformation and <20% content of α‐helical conformation, with the α‐helical segment placed near the N‐terminus. Finally, similarity searching and topology‐based structure prediction underline a significant sequential and structural similarity between the conserved N‐terminal domain of cathelicidins and cystatin‐like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki= 0.6–3 μM).
Analysis of the Specificity of the AMP‐Activated Protein Kinase by Site‐Directed Mutagenesis of Bacterially Expressed 3‐hydroxy 3‐methylglutaryl‐CoA Reductase, Using a Single Primer Variant of the Unique‐site‐elimination Method
Authors: Ching Yick Pang , Davies Stephen P. , Hardie D. Grahame
Published: August 2004

The specificity of protein kinases is usually examined using synthetic peptide substrates, either designed variants, or, more recently random peptide libraries. However not all protein kinases utilize synthetic peptides efficiently as substrates. Even among those that do, these approaches neglect effects caused by three‐dimensional protein conformation, or the existence of determinants remote from the phosphorylation site. To follow up our previous peptide studies on the specificity of the AMP‐activated protein kinase (AMPK) [Dale, S., Wilson, W. A., Edelman, A. M., & Hardie, D. G. (1995) FEBS Lett. 361, 191–195], we have expressed the C‐terminal, catalytic domain of Chinese hamster hydroxymethylglutaryl‐CoA reductase in Escherichia coli. The domain was expressed with an N‐terminal His6 tag which allowed rapid purification on Ni2+‐agarose. The purified protein retained full enzymic activity, and, as with the native enzyme, was totally inactivated by phosphorylation by AMPK at a single site corresponding to Ser871. Using a novel modification of the unique‐site elimination method (which allowed direct mutagenesis of the double‐stranded expression vector using a single oligonucleotide primer) we expressed 18 mutations involving residues around Ser871. The results broadly confirmed the recognition motif previously proposed on the basis of peptide studies. Three of the mutants were better substrates for AMPK than the wild type, and one of these (K872A) had hydroxymethylglutaryl‐CoA reductase kinetic parameters virtually indistinguishable from the wild type. This suggests that hydroxymethylglutaryl‐CoA reductase may have been selected to be a sub‐optimal substrate for AMPK.
Sarco(Endo)Plasmic Reticulum Ca2+ Pump and Metabolic Enzyme Expression in Rabbit Fast‐type and Slow‐type Denervated Skeletal Muscles
Authors: Nozais Muriel , LomprÉ Anne‐Marie , Janmot Chantal , D'Albis Anne
Published: August 2004

Recent reports by d'Albis et al. have shown that denervation of 8‐day‐old rabbit fast‐twitch muscle (gastrocnemius) leads to the transformation of the muscle towards a slow phenotype but the changes towards slow‐type myosin isoforms and contractile properties of the muscle were temporally uncoordinated. We analysed the time course of the effects of denervation of the gastrocnemius on the expression of the sarcoplasmic reticulum calcium pump isoforms (SERCA) and on the metabolic state of the muscle. Northern‐blot analysis showed a rapid loss of the fast Ca2+ pump isoform (SERCA 1) mRNA from the denervated gastrocnemius which became of the oxidative type. The changes observed were complete as early as 35 days post‐natal, i.e at the time when changes in contractile properties were previously observed. Denervation of the slow‐twitch soleus led to a 50% decrease in the level of the slow Ca2+ pump isoform (SERCA 2) mRNA and was without effect on the metabolic state of the muscle.

These findings extend previous results suggesting that in rabbit, continuous innervation is required for differentiation of fast‐twitch muscles but is not an absolute requirement for differentiation of the slow‐twitch muscle.
Solution 1H‐NMR Structure of the Heme Cavity in the Low‐Affinity State for the Allosteric Monomeric Cyano‐Met Hemoglobins from Chironomus thummi thummi
Authors: Zhang Wei , La Mar Gerd N. , Gersonde Klaus
Published: August 2004

Solution 1H‐NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S= 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82–94]. The crystal structure for the low‐pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientations, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309–338]. All heme pocket residues for the low‐pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic‐induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.
Cys5 and Cys214 of NAD(P)H:Flavin Oxidoreductase from Escherichia coli are Located in the Active Site
Authors: Fieschi Franck , Nivière Vincent , Fontecave Marc
Published: August 2004

The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH. A sequential ordered mechanism, with NADPH binding first, operates. Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N‐ethylmaleimide (MalNEt). Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved. Altogether this suggested a crucial role of cysteine residues for catalysis. The three cysteine residues were mutated to serine residues. Single‐mutant and double‐mutant enzymes were as active as the wild‐type and Km values for both substrates remained the same. Cysteine residues are thus not important for activity. Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation. This strongly suggested that Cys5 and Cys214 are located at the flavin‐binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH.