bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular Stresses and Cytokines Activate Multiple Mitogen‐Activated‐Protein Kinase Kinase Homologues in PC12 and KB Cells
Authors: Meier Roger , Rouse John , Cuenda Ana , Nebreda Angel R. , Cohen Philip
Published: August 2004

The identities of the upstream activators of the mitogen‐activated protein (MAP) kinase homologues termed stress‐activated‐protein (SAP) kinase‐I (also known as JNK or SAPK) and SAP kinase‐2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase‐I (SAPKK‐1) and SAPKK‐2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK‐1 was capable of activating both SAP kinase‐1 and SAP kinase‐2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK‐2 activated SAP kinase‐2, but not SAP kinase‐1 in vitro. In KB cells, five distinct upstream activators of SAP kinase‐1 and SAP kinase‐2 were induced, namely SAPKK‐1, SAPKK‐2, SAPKK‐3, SAPKK‐4 and SAPKK‐5, whose appearance depended on the nature of the stimulus. SAPKK‐3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL‐1), accounted for about 95% of the SAP kinase‐2 activator activity in these cells, did not activate SAP kinase‐1 and eluted from Mono S at a lower salt concentration than SAPKK‐2. SAPKK‐4 and SAPKK‐5 were also eluted from Mono S at higher NaCl concentrations than SAPKK‐3 and these enzymes activated SAP kinase‐1 but not SAP kinase‐2. SAPKK‐4 was the only SAP kinase‐1 activator induced by interleukin‐1 or ultraviolet irradiation, while two SAP kinase‐1 activators, SAPKK‐1 and SAPKK‐5, were induced by osmotic shock or anisomycin. SAPKK‐2, SAPKK‐3, SAPKK‐4 and SAPKK‐5 were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognized by anti‐MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine‐stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.
Mechanisms by Which Fatty‐Acyl‐CoA Esters Inhibit or Activate Glucose‐6‐Phosphatase in Intact and Detergent‐Treated Rat Liver Microsomes
Authors: Mithieux Gilles , Zitoun Carine
Published: August 2004

We have studied the effects of fatty‐acyl‐CoA esters on the activity of Slucose‐6‐phosphatnse (Glc6Pase) in untreated and detergent‐treated liver microsomes. Fatty‐acyl‐CoA esters with chain lengths less than or equal to nine carbons do not inhibit Glc6Pase. Medium‐chain fatty‐acyl‐CoA esters (10–14 carbons) inhibit Glc6Pase of untreated microsomes in a dose‐dependent manner in the range 1‐20 μM. The inhibitory effect is also dependent on the acyl‐chain length. The higher the chain length, the stronger the inhibitory effect. It is also dependent on the microsomal protein concentration. The higher the protein concentration, the lower the inhibitory effect. Fatty‐acyl‐CoA esters with longer chain length (equal lo or higher than 16 carbons) inhibit Glc6Pase of untreated microsomes within the range 1–2 μM. However, the inhibitory effect is cither partially or totally cancelled, or even changed into an activation effect at higher concentrations. This is due to the release of mannose‐(6‐phosphataAe latency. The inhibition is fully reversible in the presence of bovine serum albumin. The mechanism of the Glc6Pase inhibition in Untreated microsomes is uncompetitive (Ki for myristoyl‐CoA = 1.2±0.3 μM, mean±SD, n= 3). Glc6Pase of detergent‐treated microsomes is also inhibited by fatty‐acyl‐CoA esters, albeit less efficiently. In this case, the mechanism is non‐competitive (Kifor myristoyl‐CoA = 29±3 μM).
Aminopeptidase from Streptomyces griseus
Authors: Maras Bruno , Greenblatt Harry M. , Shoham Gil , Spungin‐Bialik Anya , Blumberg Shmaryahu , Barra Donatella
Published: August 2004

The aminopeptidase from Streptomyces griseus is a calcium‐activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein. This aminopeptidase rapidly hydrolyzes peptide bonds formed by N‐terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine. We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da. A search in the Swiss‐Prot database for sequence similarities revealed a low degree of identity (26–34%) to Saccharomyces cerevisiae aminopeptidase Y, Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5‐kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family. In all these proteins, the residues that are known to be involved in zinc coordination are conserved.
Distinct Exocytotic Responses of Intact and Permeabilised Chromaffin Cells After Cleavage of the 25‐kDa Synaptosomal‐Associated Protein (SNAP‐25) or Synaptobrevin by Botulinum Toxin A or B
Authors: Lawrence Gary W. , Foran Patrick , Dolly J. Oliver
Published: August 2004

Botulinum neurotoxin (BoNT) types A and B are Zn2+‐requiring endoproteases which potently block neurotransmitter release by cleavage of a 25‐kDa synaptosomal‐associated protein (SNAP‐25) and synaptobrevin, respectively. Synaptobrevin is important for the exocytosis of catecholamines from dense‐core granules and evidence is presented here for the involvement of SNAP‐25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by Ba2+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP‐25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP‐25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP‐25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20 μM Ca2+ (and to a lesser extent by 1 mM Ba2+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg · ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+‐stimulated or Ba2+‐stimulated BoNT/A‐resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNT/E to abolish secretion from permeabilised cells and to delete 26 amino acids from the C‐terminus of SNAP‐25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP‐25 with other proteins involved in regulated exocytosis.
Solution Structure of the DNA‐Binding Domain of the Tomato Heat‐Stress Transcription Factor HSF24
Authors: Schultheiss Jürgen , Kunert Olaf , Gase Uwe , Scharf Klaus‐Dieter , Nover Lutz , Rüterjans Heinz
Published: August 2004

Two‐dimensional‐NMR and three‐dimensional‐NMR experiments were performed to determine the solution structure of the DNA‐binding domain of the tomato heat‐stress transcription factor HSF24. Samples of uniformly 15N‐labeled and 15N, 13C‐labeled recombinant proteins were used in the investigation. A near‐complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three‐dimensional triple‐resonance experiments, whereas three‐dimensional 15N‐TOCSY‐heteronuclear‐single‐quantum‐correlation‐spectroscopy, HCCH‐COSY and HCCH‐TOCSY spectra were recorded for side‐chain assignments. 885 non‐redundant distance constraints from two‐dimensional‐homonuclear and three‐dimensional‐15N‐edited and 13C‐edited NOESY spectra and 40 hydrogen‐bond constraints from exchange experiments were used for structure calculations. The resulting three‐dimensional structure contains a three‐helix bundle and a small four‐stranded antiparallel β‐sheet that forms a hydrophobic core. The two C‐terminal helices are parts of a highly conserved helix‐turn‐helix motif that is probably involved in DNA recognition and binding. In contrast to heat‐stress factors from yeast and animals, the plant heat‐stress factors lack a loop of 11 amino acid residues inserted between β3 and β4. This leads to a tight turn between these β‐strands.
A Mechanism for Iron Uptake by Transferrin
Authors: Pakdaman Rowchanak , Chahine Jean‐Michel El Hage
Published: August 2004

Iron uptake by transferrin from iron nitrilotriacetate (FeNAc3,) in the presence of bicarbonate has been investigated in the pH range 6.5–8. Apotransferrin, in interaction with bicarbonate, extracts iron from FeNAc3, without the formation of an intermediate protein‐iron‐ligand mixed complex (iron‐exchange‐equilibrium constant, K1, =1 ± 0.05; direct second‐order‐rate constant, k1= 8.0 × 104± 0.5 × 104 M−1 s−1; reverse second‐order‐rate constant, k−1, = 7.5 × 104± 0.5 × 104 M−1 s−1. The newly formed iron‐protein complex loses a single proton (proton‐dissociation constant, Ka= 16 ± 1.5 nM) and then undergoes a modification of its conformation followed by loss of two or three protons (first‐order‐rate constant, k2= 2.80 ± 0.10 s−1). This induces a new modification in the conformation (first‐order‐rate constant, k3= 6.2 × 10−2± 0.3 × 10−2 s−1). This second modification in conformation controls the rate of iron uptake by the N‐site of the protein and is followed by loss of one proton (K3a, = 6.80 nM). Finally, the holoprotein or the monoferric transferrin in its final equilibrated state is produced by a third modification in the conformation that occurs after approximately 3000 s. Iron uptake by the N‐site does not occur when the apotransferrin interacts with bicarbonate. Nevertheless, it occurs with the monoferric transferrin, in which iron is bound to the C‐site, in its final state of equilibrium by a mechanism similar to that of iron uptake by the C‐site of apotransferrin. These modifications in the conformation of the protein occur after iron uptake by the C‐site and may be important for the recognition of the protein by its receptor prior to iron delivery by endocytosis.
Half‐of‐the‐sites Reactivity of Bovine Serum Amine Oxidase
Authors: De Biase Daniela , Agostinelli Enzo , DE Matteis Giovanna , Mondovì Bruno , Morpurgo Laura
Published: August 2004

The organic cofactor of bovine serum amine oxidase was identified as 2,4,5‐trihydroxyphenylalanine quinone by means of the phenylhydrazine adduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burligame, A. L. & Klinman, J. P. (1990) Science 248, 981–987]. A still debated question is, however, whether the dimeric protein binds two mol phenylhydrazine/mole or only one, that is whether it actually contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions with phenylhydrazine and other inhibitors such as semicarbazide and p‐pyridine‐2‐yl‐phenylacetohydrazide. The two latter reagents were found to bind in two steps, one mole/mole dimer in the first step with loss of catalytic activity but only about (0.10–0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or by reverse phase HPLC of the labelled peptides produced on proteolysis. Irrespective of the inhibitor nature and reacted amount, all adducts formed on proteolysis a single labelled peptide, of same 25‐amino‐acid composition, showing that the same cofactor is present in both subunits, in the same stretch of the polypeptide chain. The slow reaction of the second cofactor may be related to slow conformational equilibria, which are established after the first cofactor has reacted and are probably mediated by a change of the hydrogen bond pattern. The conformers spectroscopic properties suggest that they differ in whether the cofactor does or does not directly interact with copper.
The Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair
Authors: Lemaire Martine , Miginiac‐Maslow Myroslawa , Decottignies Paulette
Published: August 2004

Plant chloroplastic NADP–malate dehydrogenase is unique among malate dehydrogenases because of its reductive activation in the light and cofactor specificity. In this paper, the role of His229 in sorghum leaf protein has been investigated by site‐directed mutagenesis. His229 was replaced by Asn and Gln, both mutations yielding an inactive protein. The role of a conserved Asp (Asp201) as a possible partner of His229 in catalysis has been studied by the same approach. Both Asp mutants (D201A, D201N) were only slightly active and were essentially characterized by a dramatically increased Km for oxaloacetate (45–80‐fold). pH dependence of catalytic rates revealed differences between the two Asp mutants. These results demonstrate that, in sorghum leaf NADP‐dependent malate dehydrogenase, His229 is involved in catalysis in interaction with Asp201.
Cloning, Sequencing and Expression Analysis of Mouse Cartilage Matrix Protein cDNA
Authors: Aszódi Attila , Hauser Nik , Studer Daniel , Paulsson Mats , Hiripi László , Bósze Zsuzsa
Published: August 2004

A cDNA encoding the mouse cartilage matrix protein (CMP) was cloned following the reverse‐transcription polymerase chain reaction and rapid amplification of cDNA ends procedures using mRNA isolated from trachea. The open reading frame encodes a product of 500 amino acids. Large parts of the protein have been completely conserved when compared to chicken and human sequences, including all 12 cysteine residues of the mature CMP. In situ hybridization reveals an even distribution of the CMP mRNA in the developing skeleton, which is followed by a zonal distribution paralelling hypertrophy and calcification. From early cartilage differentiation and onwards, CMP transcript is absent in the forming articular surfaces and intervertebral discs. Extraskeletal expression of CMP mRNA was detected in the adult eye.
Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis
Authors: Cesura Andrea M. , Gottowik Jürgen , Lahm Hans‐Werner , Lang Gabrielle , Imhof René , Malherbe Pari , Röthlisberger Urs , Prada Mosé Da
Published: August 2004

The structural features of the active site of human monoamine oxidase B (MAO‐B) were investigated by affinity labeling and site‐directed mutagenesis. The pseudosubstrate inhibitor N‐[2‐aminoethyl]‐5‐chloro‐2‐pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO‐B by reduction of the enzyme‐inhibitor complex with NaBH3CN. Analysis of the flavin spectrum of [3H] lazabemide‐labeled human MAO‐B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO‐B. These results indicate that [3H] lazabemide is incorporated into the MAO‐B peptide stretch containing the FAD‐modified Cys397. The function of putative active‐site residues contained in this region was investigated by site‐directed mutagenesis and expression of the mutant proteins in HEK‐293 cells. Substitution of His382 of MAO‐B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO‐B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.‐F., Chen, K. & Shih, J. C. (1993) Mol. Pharmacol. 43, 888–893], the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO‐B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity.
Isolation, Characterization and Electron Microscopy Analysis of A Hemidiscoidal Phycobilisome Type from the Cyanobacterium Anabaena sp. PCC 7120
Authors: Ducret Axel , Sidler Walter , Wehrli Ernst , Frank Gerhard , Zuber Herbert
Published: August 2004

In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus. Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathway along the antenna. Upon dissociation, the phycobiliproteins were released from the phycobilisome. One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion‐exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE. The polypeptides contained in the phycobilisome of Anabaena sp. PCC 7120 were subjected to SDS/PAGE, blotted onto poly(vinylidendifluoride) membranes and identified by amino‐terminal sequence analysis. The amino‐terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes. Partial amino‐terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here. Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp. PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria.

The phycobilisome of Anabaena sp. PCC 7120 was extensively analyzed by electron microscopy. It differs from the common hemidiscoidal tricylindrical, six‐rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner. One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each. On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp. PCC 7120, M. laminosus and Anabaena variabilis is proposed. This model accounts more accurately for the ‘grape’ pattern typically exhibited by these phycobilisomes in electron micrographs.
Guanine‐Rich Oligonucleotides Targeted to a Critical R · Y Site Located in the Ki‐ras Promoter
Authors: Alunni‐Fabbroni Marianna , Manzini Giorgio , Quadrifoglio Franco , Xodo Luigi E.
Published: August 2004

The promoter of the murine Ki‐ras proto‐oncogene contains a (C+G)‐rich homopurine · homopyrimi‐dine (R · Y) sequence that is essential for transcription activity. We have designed two G‐rich oligonucleotides, d(TGGGTGGGTGGTTGGGTGGG) (20GT) and d(AGGGAGGGAGGAAGGGAGGG) (20AG), that have the potential to bind the critical Ki‐ras sequence via triplex‐helix formation. Band‐shift experiments have shown that 20GT binds the Ki‐ras R · Y duplex with a ΔG value of ‐40 ± 5 kJ/mol, while 20AG appeared to have a lower affinity under the experimental conditions adopted: 50 mM Tris/HCl, pH7.4, 50 mM NaCl, 5 mM MgCl2, 25°C. In the absence of Mg2+, 20GT did not bind to the Ki‐ras R · Y target, while 20AG exhibited the same affinity observed in the magnesium‐containing buffer. To gain insight into the solution properties of 20GT and 20AG, we have performed several experiments including polyacrylamide gel electrophoresis (PAGE), hydroxyapatite chromatography, ultraviolet absorption melting and circular dichroism (CD). We found that 20AG rapidly self‐associates into presumably a duplex, even at low concentration (<1 μM), while 20GT forms aggregates slowly, a process favoured by high oligonucleotide concentrations (>25 μM). The critical Ki‐ras sequence was inserted in Bluescript KS +, downstream from the T7 promoter, to investigate to what extent 20AG and 20GT, which are directed against the R · Y target, are able to inhibit T7 RNA polymerase transcription, under near‐physiological conditions. Transcription experiments conducted in vitro at pH 7.4 have shown that oligonucleotide 20GT produced a remarkable repression of T7 RNA polymerase activity in the concentration range (10–25 μM), whereas 20AG had little effect on transcription. In conclusion, the results of this work together with other data reported in the literature [Olivas, W. M. & Maher, L. J. Ill (1995) Biochemistry 34, 278–284; Noonberg, S. B., Francois, J.‐C, Garestier, T. & Helene, C. (1995) Nucleic Acids Res. 23, 1956–1963], demonstrate that G‐rich oligonucleotides, in particular (G, A)‐sequences, may raise problems for in vivo application due to self‐aggregation.
Complete cDNA Sequence of the Preproform of Human Pregnancy‐Associated Plasma Protein‐A
Authors: Haaning Jesper , Oxvig Claus , Overgaard Michael T. , Ebbesen Peter , Kristensen Torsten , Sottrup‐Jensen Lars
Published: August 2004

A cDNA that encodes the prepropeptide of pregnancy‐associated plasma protein‐A (preproPAPP‐A), a putative metalloproteinase, has been cloned and sequenced. PAPP‐A is synthesized in the placenta as a 1627‐residue precursor preproprotein with a putative 22‐residue signal peptide and a highly basic propeptide of 58 residues. The preproPAPP‐A–encoding transcript contains a region with an extremely high G+C content and has an unusually long 5′ untranslated region with several upstream short ORF. No alternatively spliced products could be identified by means of Northern blotting experiments or with rapid amplification of 5′ cDNA ends experiments. A stretch within the 5′ untranslated region shows sequence identities to a partial cDNA isolated from brain and a to cAMP‐inducible sequence from a choriocarcinoma cell line.
Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA
Authors: Fletcher Michael C. , Fox Keith R.
Published: August 2004

We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT)n than An· Tn. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC)n· (GT)n present good binding sites; in this context, dissociation from CGCG is faster than from TGCA.
Purification and Characterization of a 5′ to 3′ Exoribonuclease from Rabbit Reticulocytes that Degrades Capped and Uncapped RNAs
Authors: Somoskeöy Szabolcs , Rao Manjunath N. , Slobin Lawrence I.
Published: August 2004

The cytoplasm of mammalian cells undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5′ to 3′ manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat‐labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10–200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5′ end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate‐terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate‐limiting step involves the positioning of the enzyme at the 5′ end of the substrate and/or the cleavage of the first internucleotide bond.
The Inhibition of PhospoEnol Pyruvate Carboxykinase Following in Vivo Chronic Phenobarbital Treatment in the Rat is Due to a Post‐Translational Event
Authors: Chauvin Christiane , Brilloit‐Petit Christine , Argaud Doriane , Catelloni Franoise , Velours Jean , Leverve Xavier M.
Published: August 2004

Chronic treatment of rats with phenobarbital has been reported to decrease gluconeogenesis in rat hepatocytes by a 50% inhibition of phosphoenolpyruvate (P‐pyruvate) carboxykinase activity [Argaud, D., Halimi, S., Catelloni, F. & Leverve, X. (1991) Biochem. J. 280, 663–669]. Contrary to the current knowledge of P‐pyruvate carboxykinase regulation, we failed to find a diminution of either P ‐pyruvate carboxykinase protein (by using a polyclonal antibody) or P ‐pyruvate carboxykinase mRNA, in the liver of rats treated with phenobarbital for 2 weeks. Kinetic studies of P ‐pyruvate carboxykinase activity, measured by either carboxylation of P ‐pyruvate or decarboxylation of oxaloacetate, revealed a decrease in both Vmax and Km after phenobarbital treatment, whereas the nutritional state affected only the Vmax, as expected. Assessment of P ‐pyruvate carboxykinase specificity was confirmed by the full inhibition of the enzyme with its specific inhibitor 3‐mercaptopicolinate in the micromolar range. P ‐Pyruvate carboxykinase, purified either by ammonium sulfate fractionation or by immunoprecipitation, exhibited a similar decrease in affinity after phenobarbital treatment. Although the molecular mass does not appear to be altered, the pH sensitivity to 3‐mercaptopicolinate inhibition and the enzyme recovery after immunoprecipitation both seemed to be affected. This leads us to propose that the effect of chronic phenobarbital treatment on P ‐pyruvate carboxykinase activity is not the result of transcriptional regulation but is exerted at the post‐translational level.
Effect of Tumor Necrosis Factor‐α on Insulin‐Stimulated Mitogen‐Activated Protein Kinase Cascade in Cultured Rat Skeletal Muscle Cells
Authors: Begum Najma , Ragolia Louis , Srinivasan Malathi
Published: August 2004

Tumor necrosis factor‐α (TNF‐α) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate‐1. In this study, the acute effects of TNF‐α on the mitogen‐activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF‐α for 60 min blocked subsequent insulin‐induced activation of MAPK in a dose‐ and time‐dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno‐precipitation of MAPK and its upstream activator, mitogen‐activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF‐α. This effect of TNF‐α was not due to inhibition of insulin‐stimulated p21ras5–GTP loading or Raf‐1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF‐α‐induced inhibition of MAPK and restored insulin' effect on MAPK activity, while ortho‐vaNatlate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase‐1 inhibitor) and FK506 (phospha‐tase‐2B inhibitor) were ineffective. These results suggested an involvement of an okadaic‐acid‐sensitive serine/threonine phosphatase in TNF‐α‐induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF‐α on protein phosphatase‐1 (PP‐1) and protein phosphatase‐2A (PP‐2A) activities. As reported by us earlier, insulin rapidly stimulated PP‐1 and concomitantly inhibited PP‐2A activities in control cells. TNF‐α treatment blocked insulin‐induced activation of PP‐1. In contrast to PP‐1, TNF‐α caused a 60% increase in PP‐2A activity and insulin failed to prevent this TNF‐α effect. The time course of PP‐2A activation by TNF‐α preceded the kinetics of inhibition of MAPK. Cell‐permeable ceramide analogs mimicked the TNF‐α effect on MAPK inhibition and PP‐2A activation. We conclude that TNF‐α abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
Structure of the O‐Specific Polysaccharide of Acinetobacter baumannii O5 Containing 2‐Acetamido‐2‐Deoxy‐d‐Galacturonic Acid
Authors: Haseley Simon R. , Wilkinson Stephen G.
Published: August 2004

A polysaccharide containing 2‐acetamido‐2‐deoxy‐d‐glucose (GlcNAc), 2‐acetamido‐2‐deoxy‐l‐fucose (FucNAc), and 2‐acetamido‐2‐deoxy‐d‐galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid‐free, isolated cell walls of the reference strain for Acinetobacter baumannii sero‐group O5, by mild acid hydrolysis of the extract and chromatography of the water‐soluble products on Sephadex G‐50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide:
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Characterization of α‐Neurotoxin and Phospholipase A2 Activities from Micrurus Venoms
Authors: Rosso Jean‐Pierre , Vargas‐Rosso Orietta , Gutiérrez José María , Rochat Hervé , Bougis Pierre E.
Published: August 2004

New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse‐phase high‐performance liquid chromatography and specific assays for α‐neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven α‐neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major α‐neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short‐chain α‐neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.
Glucose uptake in Trypanosoma vivax and Molecular Characterization of its Transporter Gene
Authors: Waitumbi John Njenga , Tetaud Emmanuel , Baltz Théo
Published: August 2004

A gene, TvHT1, encoding a glucose transporter protein, has been cloned from the haemoflagellate protozoon, Trypanosoma vivax, which has an active Kreb's cycle in the mammalian stage. The deduced polypeptide is similar in amino acid sequence to other kinetoplastid hexose transporters from Trypanosoma brucei (THT1 and THT2), Trypanosoma cruzi (TcrHT1) and Leishmania (Pro‐1). The similarity is higher with THT2 (expressed in T. brucei insect forms) than with the other isoforms. The kinetic properties of glucose uptake in Chinese Hamster Ovary (CHO) cells expressing TvHT1 and in trypanosomes show a saturable transport mechanism typical of a facilitated carrier system, with a similar affinity for d‐glucose as that of the T. brucei bloodstream form carrier, THT1 (Km= 0.548 ± 0.01 mM, Vmax= 4.26 ± 0.12 nmol · min−1· mg protein−1 in CHO cells and Km= 0.585 ± 0.068 mM, Vmax= 88.5 ± 6.2 nmol · min−1· mg protein−1 in T. vivax). The specificity of the TvHT1 protein for various d‐glucose analogues, as judged by inhibition of 2‐deoxy‐d‐arabinose‐hexose transport, shows properties that are intermediate between those of THT1 on the one hand and TcrHT1 and THT2 on the other. As with the hexose transporters in the other members of Kinetoplastida, the TvHT1‐encoded system differs from erythrocyte‐type glucose transport by its moderate sensitivity to cytochalasin B and its capacity to transport fructose.
Colocalization of Cytosolic Phospholipase A2, 5‐Lipoxygenase, and 5‐Lipoxygenase‐Activating Protein at the Nuclear Membrane of A23187‐Stimulated Human Neutrophils
Authors: Pouliot Marc , Mcdonald Patrick P. , Krump Eric , Mancini Joseph A. , Mccoll Shaun R. , Weech Philip K. , Borgeat Pierre
Published: August 2004

The distribution of cytosolic phospholipase A2 (cPLA2), arachidonate 5–lipoxygenase, and 5–lipoxygenase‐activating protein (5–LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA2 and 5–lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A23187, both proteins accumulated primarily in nuclei‐containing fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5‐lipoxygenase and cPLA2 were localized in a fraction that contained nuclear membranes. In comparison, 5‐LAP was localized to the nuclear‐membrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffin‐ity labeling. In agreement with the immunoblot data, A23187 stimulation markedly enhanced 5‐lipoxygenase enzymatic activity in the nuclear‐membrane fraction, which was accompanied by decreased cytosolic 5‐lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA2, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.
Structural Studies of the Putative O‐Specific Polysaccharide of Acinetobacter baumannii O11
Authors: Haseley Simon R. , Wilkinson Stephen G.
Published: August 2004

A major polysaccharide containing d‐galactose, d‐glucose and 2‐acetamido‐2‐deoxy‐d‐galactose was obtained after mild acid hydrolysis of the water‐soluble material released by treatment of cell walls from Acinetobacter baumannii strain Oil with hot, aqueous phenol. By means of NMR studies, Smith degradation and N‐deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2‐aceta‐mido‐2‐deoxyglucose and 2‐acetamido‐2‐deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O‐antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that Oil specificity was conferred by the major polymer.
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DNA‐replication Fidelity, Mismatch Repair and Genome Instability in Cancer Cells
Authors: Umar Asad , Kunkel Thomas A.
Published: August 2004

It has been suggested that an early event in the multistep progression of a normal cell to a tumor cell could be a defect that leads to an elevated mutation rate, thus providing a pool of mutants upon which selection could act to yield a tumor. Such a mutator phenotype could result from a defect in any of several DNA transactions, including those that determine the DNA replication error rate or the ability to correct replication errors. Recent evidence for the latter is the mutator phenotype observed in tumor cells of patients having a hereditary form of colon cancer. These patients have a germline mutation in genes required for post‐replication DNA mismatch repair. A second mutation arises somatically, yielding a greatly elevated mutation rate due to an inability to correct DNA replication errors. This connection between cancer, DNA replication errors and defective mismatch repair is the subject of this review, wherein we consider the key steps and principles for high fidelity replication and how their perturbation results in genome instability.
A Class of Highly Potent Antibacterial Peptides Derived from Pardaxin, A Pore‐Forming Peptide Isolated from Moses Sole Fish Pardachirus marmoratus
Authors: Oren Ziv , Shai Yechiel
Published: August 2004

Pardaxin, a 33‐amino‐acid pore‐forming polypetide toxin isolated from the Red Sea Moses sole Pardachirus marmoratus, has a helix‐hinge‐helix structure. This is a common structural motif found both in antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Herein we show that pardaxin possesses a high antibacterial activity with a significantly reduced hemolytic activity towards human red blood cells (hRBC), compared with melittin. Its potency is comparable to that of other known native antibacterial peptides such as magainin, cecropins and dermaseptins. To determine the structural features responsible for the selective hemolytic and antibacterial activities, and the structural requirements for a high antibacterial activity, 8 truncated and modified pardaxin analogues were synthesized and structurally and functionally characterized. Each peptide was synthesized with a free carboxylate or amino group (i.e., aminated form) at its C‐terminus. The aminated form of pardaxin has both high hemolytic and antibacterial activity. A truncated analogue, with 11 amino acids removed from the C‐terminal domain, had dramatically reduced hemolytic activity. However, the aminated form of this analogue was significantly more potent than pardaxin against most bacteria tested, suggesting that the C‐terminal tail of pardaxin is responsible for non‐selective activity against erythrocytes and bacteria. Furthermore, a positive charge added to its N‐terminus significantly increased its antibacterial activity and abolished its low hemolytic activity. The 22‐amino‐acid C‐terminal domain and the short 11‐amino‐acid N‐terminal domain were, in their aminated forms, active only against gram‐positive bacteria. Secondary‐structure determination using circular dichroism spectroscopy revealed that all the aminated analogues had 25–80% more α‐helical content in 40% CF3CH2OH/water than their non‐aminated forms. Using model phospholipid membranes it was found that all the analogues that were less hemolytic but had retained antibacterial activity could permeate acidicly charged phospholipid vesicles better than zwitterionic phospholipid vesicles, a property characteristics of all the native antibacterial peptides tested so far (e.g., cecropins, magainins and dermaseptins). Pardaxin and its analogues therefore represent a new class of antibacterial peptides that can serve as a basis for the design of therapeutic agents. Furthermore, negative‐staining electron microscopy revealed that total inhibition of bacterial growth was due to total lysis of the bacterial wall. Therefore, it might be more difficult for bacteria to develop resistance to such a destructive mechanism, compared with the more specific mechanisms of the currently used antibiotics.
Inhibitory Properties of Separate Recombinant Kunitz‐Type‐Protease‐Inhibitor Domains from Tissue‐Factor‐Pathway Inhibitor
Authors: Petersen Lars C. , Bjørn Søren E. , Olsen Ole H. , Nordfang Ole , Norris Fanny , Norris Kjeld
Published: August 2004

Tissue‐factor‐pathway inhibitor (TFPT) is a multivalent inhibitor with three tandemly arranged Kunitz‐type‐protease‐inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny, W. F., Likert, K. M., Brown, S. G., Miletich, J. P. & Broze, G. J. (1989) Nature 338, 518–520] by means of site‐directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPT domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI‐(1–161)‐peptide and separate recombinant TFPI‐KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki= 250 nM), KPI domain 2 inhibited factor Xa (Ki= 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki= 0.1 nM) and chymotrypsin (Ki= 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki= 26 nM) and cathepsin G (Ki= 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three‐dimensional models of the proteins derived by comparative molecular‐modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki≈ 0.02 nM) suggested that regions other than the contact area of the KPT domain, interacted strongly with factor Xa. Secondary‐site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.
The Human Gene FALL39 and Processing of the Cathelin Precursor to the Antibacterial Peptide LL‐37 in Granulocytes
Authors: Gudmundsson Gudmundur H. , Agerberth Birgitta , Odeberg Jacob , Bergman Tomas , Olsson Berit , Salcedo Rosalba
Published: August 2004

The peptide FA‐LL‐37, previously termed FALL‐39, was originally predicted from an ORF of a cDNA clone isolated from a human bone marrow library. This peptide was synthesized and found to have antibacterial activity. We have now characterized and sequenced the complete gene for FA‐LL‐37, termed FALL39. It is a compact gene of 1963 bp with four exons. Exons 1–3 code for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Our results indicate that FALL39 is the only member of the cathelin gene family present in the human genome. Potential binding sites for acute‐phase‐response factors are identified in the promoter and in intron 2. A possible role for the cytokine interleukin‐6 in the regulation of FALL39 is discussed. Anti‐(FA‐LL‐37) IgG located the peptide in granulocytes and we isolated the mature peptide from these cells after degranulation. Structural analysis determined the mature peptide to be LL‐37. To obtain LL‐37 for antibacterial assays, synthetic FA‐LL‐37 was degraded with dipeptidyl‐peptidase I. This analysis showed that mature LL‐37 is a potent antibacterial peptide.
Pyruvate Decarboxylase from Pisum sativum
Authors: Mücke Udo , Wohlfarth Thomas , Fiedler Ulrike , Bäumlein Helmut , Rücknagel Karl Peter , König Stephan
Published: August 2004

To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X‐ray crystallography, it was necessary to elucidate the protein sequence.

Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3′‐and 5′‐rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.
Sequence‐Specific Resonance Assignments of the 1H‐NMR Spectra and Structural Characterization in Solution of the HIV‐1 Transframe Protein p6*
Authors: Beissinger Martina , Paulus Christina , Bayer Peter , Wolf Hans , Rösch Paul , Wagner Ralf
Published: August 2004

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV‐1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag‐Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX‐KG and expressed in Escherichia coli as a fusion protein with glutathione S ‐transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70‐amino‐acid polypeptide which is extended by two additional residues at the N‐terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size‐exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H‐NMR spectroscopy were applied to characterize the structure of the protein. Two‐dimensional NMR spectra provided essentially complete sequence‐specific resonance assignments at pH 5.9. Although there is evidence for a helix‐forming tendency in the N‐terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV‐1 protease in structural and functional in vitro studies.
Primary Structure, Expression and Developmental Regulation of a Dictyostelium Calcineurin A Homologue
Authors: Dammann Heike , Hellstern Simon , Husain Qayyum , Mutzel Rupert
Published: August 2004

cDNA clones for the catalytic subunit of Ca2+/calmodulin(CaM)‐dependent protein phosphatase (calcineurin A, protein phosphatase 2B) from Dictyostelium discoideum were isolated by functional screening of a λgt11 lysogen expression library with labeled Dictyostelium CaM. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N‐terminal and C‐terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shown to display similar enzymatic properties as the enzyme from other sources. On Western blots specific antibodies against the protein recognized a band of approximately 80 kDa that migrated with an endogenous CaM‐binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed.