bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant expression of N‐terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase
Authors: Ullrich Alexandra , Knecht Wolfgang , Fries Markus , Löffler Monika
Published: March 2003

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full‐length rat and human dihydroorotate dehydrogenase, here we expressed N‐terminal‐truncated C‐terminal‐histidine‐tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N‐terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal‐chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 µmol·min−1·mg−1 at pH optimum of 8.0–8.1. Flavin analyses by UV‐visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6–1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (Km = 11 µm dihydroorotate; Km = 7 µm ubiquinone) and human enzyme (Km = 10 µm dihydroorotate; Km = 14 µm ubiquinone) were very close to those recently reported for the full‐size enzymes. The constants for the mouse enzyme, Km = 26 µm dihydroorotate and Km = 62 µm ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N‐terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77‐1726, MNA715 and MNA279 with the rat and human enzyme, the N‐termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N‐terminal part of the human enzyme seemed to be of crucial importance for the ‘slow‐binding’ features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti‐proliferative and immunosuppressive action.
Structure–function relationships of hormone‐sensitive lipase
Authors: Østerlund Torben
Published: March 2003

Research into the structure–function relationships of lipases and esterases has increased significantly during the past decade. Of particular importance has been the deduction of several crystal structures, providing a new basis for understanding these enzymes. The generated insights have, together with cloning and expression, aided studies on structure–function relationships of hormone‐sensitive lipase (HSL). Novel phosphorylation sites have been identified in HSL, which are probably important for activation of HSL and lipolysis. Functional and structural analyses have revealed features in HSL common to lipases and esterases. In particular, the catalytic core with a catalytic triad has been unveiled. Furthermore, the investigations have given clear suggestions with regard to the identity of functional and structural domains of HSL. In the present paper, these studies on HSL structure–function relationships and short‐term regulation are reviewed, and the results presented in relation to other discoveries in regulated lipolysis.
Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12‐dependent glycerol dehydratase of Citrobacter freundii
Authors: Seifert Corinna , Bowien Susanne , Gottschalk Gerhard , Daniel Rolf
Published: March 2003

The coenzyme B12‐dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3‐propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell‐free extracts of a recombinant E. coli strain producing both His6‐tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably α2β2. The factor rapidly reactivated glycerol‐ or O2‐inactivated hologlycerol dehydratase and activated the enzyme–cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. The DhaF–DhaG complex and DhaF exhibited ATP‐hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF–DhaG complex of C. freundii efficiently cross‐activated the enzyme–cyanocobalamin complex and the glycerol‐inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.
Cloning and characterization of Aspergillus niger genes encoding an α‐galactosidase and a β‐mannosidase involved in galactomannan degradation
Authors: Ademark Pia , de Vries Ronald P. , Hägglund Per , Stålbrand Henrik , Visser Jaap
Published: March 2003

α‐Galactosidase (EC 3.2.1.22) and β‐mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an α‐galactosidase (AglC) and a β‐mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N‐glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. nigerα‐galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild‐type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno‐oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
Protein engineering of Bacillus megaterium CYP102
Authors: Carmichael Angus B. , Wong Luet‐Lok
Published: March 2003

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild‐type CYP102 (P450BM‐3) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40‐fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min−1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min−1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10‐phenanthrenequinone, the K‐region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.
Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.
Authors: Meskys Rolandas , Harris Richard J. , Casaite Vida , Basran Jaswir , Scrutton Nigel S.
Published: March 2003

The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1‐IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N‐terminal amino‐acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino‐acid sequence with amino‐acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P‐1. Deletion and expression analysis, and alignment of the deduced amino‐acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide‐binding, flavinylation and folate‐binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate‐dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate‐dependent enzymes.
The ABC transporter Pdr5p mediates the efflux of nonsteroidal ecdysone agonists in Saccharomyces cerevisiae
Authors: Hu Wenqi , Feng Qili , Palli Subba R. , Krell Peter J. , Arif Basil M. , Retnakaran Arthur
Published: March 2003

We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH‐5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH‐5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter‐deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Δpdr5 and Δpdr5Δsnq2) retained significantly higher levels of 14C‐radiolabeled RH‐5992 within the cells when compared to wild‐type strain or single deletion mutants of SNQ2 (Δsnq2) and YCF1 (Δycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH‐5992 from these cells as efficiently as the wild‐type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH‐5992 in yeast. This exclusion was temperature‐dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH‐0345 and [14C]RH‐2485, but not [14C]RH‐5849, indicating that these three compounds share the same transporter Pdr5p for efflux.
Oxidative and hydrolytic properties of β‐amyloid
Authors: Brzyska Maria , Bacia Andrzej , Elbaum Danek
Published: March 2003

β‐Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733–738]. In the present work we compare the ability of β‐amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF‐DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of β‐amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human β‐amyloid (1–40), containing three amino‐acid substitutions (Arg5→Gly, Tyr10→Phe and His13→Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that β‐amyloid‐mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF‐DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox‐active metal ions and was practically oxygen independent while the oxidation process was redox‐active‐metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide‐mediated singlet oxygen intermediate in this Fenton mechanism‐driven reaction. These results indicate that hydrolytic and oxidative properties of β‐amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to β‐amyloid toxicity.
Differentiation between conformational and autoproteolytic stability of the neutral protease from Bacillus stearothermophilus containing an engineered disulfide bond
Authors: Dürrschmidt Peter , Mansfeld Johanna , Ulbrich‐Hofmann Renate
Published: March 2003

The introduction of a disulfide bond into the neutral protease from Bacillus stearothermophilus by the double mutation G8C/N60C had resulted in an extremely thermostable enzyme with a half‐life of 35.9 min at 92.5 °C [Mansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., van den Burg, B., Venema, G., Ulbrich‐Hofmann, R. & Eijsink, V.G. (1997) J. Biol. Chem. 272, 11152–11156]. The study in guanidine hydrochloride of this enzyme and the respective wild‐type enzyme allowed us to distinguish between the stability toward global unfolding and autoproteolysis. At low protease concentrations (20 µg·mL−1) and short periods of incubation with guanidine hydrochloride (5 min), transition curves without the interference by autoproteolysis could be derived from fluorescence emission measurements. The effect of the disulfide bond on the global unfolding of the protein proved to be smaller than expected. In contrast, the measurement of autoproteolysis at higher protein concentrations (100 µg·mL−1) by quantitative evaluation of the bands of intact protein on SDS/PAGE revealed a strong stabilization toward autoproteolytic degradation by the disulfide bond. The rate of autoproteolysis in guanidine hydrochloride was found to be much lower than that of thermal denaturation, which can be attributed to the inhibition of the proteases by this denaturant. The results suggest that the disulfide bond stabilizes the protease against autoproteolysis more than against global unfolding. Autoproteolysis starts as soon as the cleavage sites in flexible external structural regions become accessible. It is suggested that the stabilizing effect of the disulfide bond is caused by the fixation of the crucial loop region 56–69 or by hindrance of the primary cleavage in this region by the amino acid exchanges.
Mechanism of activation of the double‐stranded‐RNA‐dependent protein kinase, PKR
Authors: Vattem Krishna M. , Staschke Kirk A. , Wek Ronald C.
Published: March 2003

An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the α subunit of eIF2. Binding of viral dsRNAs to two dsRNA‐binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein–protein interactions and that trans‐autophosphorylation is the final step in the mechanism of activation of PKR.
Biochemical characterization of nuclear pore complex protein gp210 oligomers
Authors: Favreau Catherine , Bastos Ricardo , Cartaud Jean , Courvalin Jean‐Claude , Mustonen Pekka
Published: March 2003

The membrane‐spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N‐terminal domain, a short C‐terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration. Cross‐linking of proteins in isolated membranes prior to solubilization dramatically increases the proportion of dimers. The dimers are SDS‐resistant, as previously observed for some integral membrane proteins of cis‐Golgi and plasma membrane proteins, including glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration and denaturing electrophoresis, but unlike the dimers are dissociated by reduction and heating in the presence of SDS. We propose that gp210 is organized into the pore membrane as a large array of gp210 dimers that may constitute a luminal submembranous protein skeleton.
Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site‐specific mutants
Authors: Clantin Bernard , Tricot Catherine , Lonhienne Thierry , Stalon Victor , Villeret Vincent
Published: March 2003

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 °C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild‐type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.
Identification of the β‐dystroglycan binding epitope within the C‐terminal region of α‐dystroglycan
Authors: Sciandra Francesca , Schneider Martina , Giardina Bruno , Baumgartner Stefan , Petrucci Tamara C. , Brancaccio Andrea
Published: March 2003

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post‐translationally cleaved to form two different subunits, α and β. The extracellular α‐dystroglycan is a membrane‐associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane β‐dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. α‐ and β‐Dystroglycan interact tightly but noncovalently. We have previously shown that the N‐terminal region of β‐dystroglycan, β‐DG(654–750), binds to the C‐terminal region of murine α‐dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid‐phase binding assays, the C‐terminal sequence of α‐dystroglycan containing the binding epitope for β‐dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550–585, is required for binding the extracellular region, amino acids 654–750 of β‐dystroglycan. Recently, a dystroglycan‐like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550–585 sequence of vertebrate α‐dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) β‐dystroglycan (Kd = 6 ± 1 µm). These data confirm the proper identification of the β‐dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan‐specific binding drugs, that could have important biomedical applications.
The role of Thr160 phosphorylation of Cdk2 in substrate recognition
Authors: Holmes Jennifer K. , Solomon Mark J.
Published: March 2003

Full activation of cyclin‐dependent kinases (Cdks) requires binding to a cyclin and phosphorylation on an activating site equivalent to Thr160 in Cdk2 by the Cdk‐activating kinase. Much is known about the effects of cyclin binding, but the role of the activating phosphorylation is less well understood. We have characterized the effects of Thr160 phosphorylation of Cdk2 on its interactions with substrates, particularly with the P + 3 position. We find that an ionic interaction participates in the recognition of the P + 3 position of the substrate and confirms an observation from structural studies indicating that a key element of this recognition is an interaction between the lysine at the P + 3 position and the Thr160 phosphate of Cdk2. The major effect of disrupting the lysine–phosphate interaction was on kcat values rather than Km values, suggesting that the energy from this interaction is used to align the substrate for efficient catalysis. A lack of effect of Thr160 phosphorylation on the ATPase activity of Cdk2 supported this interpretation.
Protein‐bound conformation of a specific inhibitor against Candida albicans myristoyl‐CoA:protein N‐myristoyltransferase in the ternary complex with CaNmt and myristoyl‐CoA by transferred NOE measurements
Authors: Miura Takaaki , Klaus Werner , Ross Alfred , Sakata Kiyoaki , Masubuchi Miyako , Senn Hans
Published: March 2003

Transferred nuclear Overhauser enhancement (trNOE) experiments have been performed to study the bioactive conformation(s) of Ro09‐3472/000 derivatives in the ternary complex with Candida albicans myristoyl‐CoA: protein N‐myristoyltransferase (CaNmt) and myristoylCoA (MyrCoA). A critical step in the trNOE study is to identify ‘true’ trNOEs in the spectra. Nonspecific binding of ligands to target proteins and/or spin diffusion effects can give rise to ‘false’ trNOEs, which may lead to an incorrect conclusion if used to derive bound conformations. In this study for all ligands the observed trNOEs arose from specific binding interactions with the active site of CaNmt. This was shown by displacing the ligand with the known tightly binding active‐site inhibitor 1 [Devadas, B., Zupec, M.E., Freeman, S.K., Brown, D.L., Nagarajan, S., Sikorski, J.A., McWherter, C.A., Getman, D. P. & Gordon, J.I. (1995) J. Med. Chem.38, 1837–1840] and measuring the resonance linewidths in the NMR spectrum before and after addition of the competitive inhibitor. The compounds were also tested for nonspecific protein binding with bovine serum albumin (BSA) using the same method. Of the six compounds tested, Ro09‐3700/001 (racemate) and its optically pure enantiomers, Ro09‐4764/001(S) and Ro09‐4765/001(R), showed both specific binding to CaNmt and no interaction with BSA. The NMR data of these molecules in the ternary complex with CaNmt/MyrCoA could thus be used for a detailed structural analysis. Thereby, the conformation of the bound ligand was obtained from a conformational search using the observed trNOEs as a selection filter. The NMR‐determined conformations are in good agreement with the recently solved CaNmt‐bound X‐ray structures of two similar Ro09‐3472/000 derivatives.
Functional asymmetry of the ATP‐binding‐cassettes of the ABC transporter TAP is determined by intrinsic properties of the nucleotide binding domains
Authors: Daumke Oliver , Knittler Michael R.
Published: March 2003

The ATP‐binding‐cassette (ABC) transporter associated with antigen processing (TAP) delivers peptides into the ER. TAP consists of two polypeptides (TAP1 and TAP2) each with an N‐terminal transmembrane (TMD) and a C‐terminal nucleotide binding domain (NBD). The two highly homologous NBDs of TAP show different nucleotide binding specificites, and identical mutations in the domains can have different effects on peptide transport. We asked whether this functional asymmetry of the NBDs is an intrinsic property or is imposed by the TMDs to which they are linked. To investigate the functional interdependence of the TAP domains, we created various TAP variants in which TMDs and/or NBDs were exchanged. All TAP variants except those with two TMDs of TAP1 could assemble. The TMDs did not affect the different nucleotide binding properties of the NBDs. The TAP variant with switched NBDs showed active peptide transport while the variants with pairs of identical NBDs or TMDs were inactive. Although both types of TMDs and NBDs have to be present for peptide transport they do not have to be assorted as in wild‐type TAP. Thus, TAP domains seem to preserve functional autonomy despite their fusion into single polypeptide chains. We propose that the two NBDs act as nonequivalent ‘modules’ that directly determine the functional asymmetry of the included ATP‐binding‐cassettes. This provides a new insight into the function of NBDs and opens up new possibilities to investigate the molecular mechanism of the ‘NBD engine’ in ABC transporters.
Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M‐type homopolymer
Authors: Mignogna Guiseppina , Chiaraluce Roberta , Consalvi Valerio , Cavallo Stefano , Stefanini Simonetta , Chiancone Emilia
Published: March 2003

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is composed of a single subunit that contains both the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleation site, typical of mammalian L chains. Comparison of the amino‐acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M‐type homopolymer. Interestingly, the T. bernacchii ferritin chain shows 85.7% identity with a cold‐inducible ferritin chain of the rainbow trout Salmo gairdneri.

The structural and functional properties indicate that cold acclimation and functional adaptation to low temperatures are achieved without significant modification of the protein stability. In fact, the stability of T. bernacchii ferritin to denaturation induced by acid or temperature closely resembles that of mesophilic mammalian ferritins. Moreover iron is taken up efficiently and the activation energy of the reaction is 74.9 kJ·mol−1, a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJ·mol−1).
Characterization of the self‐splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes
Authors: Haugen Peik , De Jonckheere Johan F. , Johansen Steinar
Published: March 2003

The two group I introns Nae.L1926 and Nmo.L2563, found at two different sites in nuclear LSU rRNA genes of Naegleria amoebo‐flagellates, have been characterized in␣vitro. Their structural organization is related to that of the mobile Physarum intron Ppo.L1925 (PpLSU3) with ORFs extending the L1‐loop of a typical group IC1 ribozyme. Nae.L1926, Nmo.L2563 and Ppo.L1925 RNAs all self‐splice in vitro, generating ligated exons and full‐length intron circles as well as internal processed excised intron RNAs. Formation of full‐length intron circles is found to be a general feature in RNA processing of ORF‐containing nuclear group I introns. Both Naegleria LSU rDNA introns contain a conserved polyadenylation signal at exactly the same position in the 3′ end of the ORFs close to the internal processing sites, indicating an RNA polymerase II‐like expression pathway of intron proteins in vivo. The intron proteins I‐NaeI and I‐NmoI encoded by Nae.L1926 and Nmo.L2563, respectively, correspond to His‐Cys homing endonucleases of 148 and 175 amino acids. I‐NaeI contains an additional sequence motif homologous to the unusual DNA binding motif of three antiparallel β sheets found in the I‐PpoI endonuclease, the product of the Ppo.L1925 intron ORF.
Drug recognition of a DNA single strand break
Authors: Williams Huw E. L. , Colgrave Michelle L. , Searle Mark S.
Published: March 2003

Two DNA hairpin motifs (5′‐GCGAAGC‐3′ and 5′‐ACGA AGT‐3′), both stabilized by a 5′‐GAA loop, have been used to design novel intramolecular double hairpin structures (5′‐GCGAAGCACGAAGT‐3′ and 5′‐ACGAAGTGCG AAGC‐3′) in which coaxial stacking of the two hairpin components generates a double‐stranded stem region effectively with a single‐strand break in the middle of the sequence at either the TG or CA step between unconnected 3′ and 5′ terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5′‐TG (5′‐CA) site formed by the mutually stacked hairpins despite the break in the sugar‐phosphate backbone on one strand. The complex resembles the structure of nogalamycin–DNA complexes with the drug bound at 5′‐TG sites in intact duplex sequences, with π‐stacking interactions probably the single dominant stabilizing interaction.
Structures of two histidine ammonia‐lyase modifications and implications for the catalytic mechanism
Authors: Baedeker Mathias , Schulz Georg E.
Published: March 2003

Histidine ammonia‐lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the α‐amino group of histidine using a 4‐methylidene‐imidazole‐5‐one (MIO), which is formed autocatalytically from the internal peptide segment 142Ala‐Ser‐Gly. The structure of the enzyme inhibited by a reaction with l‐cysteine was established at the very high resolution of 1.0 Å. Five active center mutants were produced and their catalytic activities were measured. Among them, mutant Tyr280→Phe could be crystallized and its structure could be determined at 1.7 Å resolution. It contains a planar sp2‐hybridized 144‐N atom of MIO, in contrast to the pyramidal sp3‐hybridized 144‐N of the wild‐type. With the planar 144‐N atom, MIO assumes the conformation of a putative intermediate aromatic state of the reaction, demonstrating that the conformational barrier between aromatic and wild‐type states is very low. The data led to a new proposal for the geometry for the catalyzed reaction, which also applies to the closely related phenylalanine ammonia‐lyase (EC 4.3.1.5). Moreover, it suggested an intermediate binding site for the released ammonia.
Differences in Timidity and Escape Responses between Predator‐naive and Predator‐sympatric Rainbowfish Populations
Authors: Brown Culum , Warburton Kevin
Published: March 2003

Responses of rainbowfish (Melanotaenia duboulayi) from two populations towards a) an active and a passive predatory fish and b) a novel trawl apparatus, were compared. Predator‐sympatric fish avoided the fish predators and showed stronger avoidance behaviour in response to the active predator. These fish used predator inspection excursions to rapidly assess the potential risk and their escape responses were consistently effective. In contrast the predator‐naive fish ignored the passive predator but were continually drawn towards the active predator possibly due to generalized curiosity and the absence of significant negative feedback from the predator, which was restrained by a clear Perspex partition. Despite this attraction, the predator‐naive fish did not display typical predator inspection behaviour and showed very poor escape performance when initially confronted by the trawl apparatus. Many of these fish, however, showed rapid improvement in their escape performance through learning. These results suggest that predator‐sympatric rainbowfish have the capacity to assess the level of threat posed by a predator and predator‐naive rainbowfish learn to implement appropriate escape strategies when forced to evade a threat.
Sleeping Site Selection by the Golden‐handed Tamarin Saguinus midas midas: The Role of Predation Risk, Proximity to Feeding Sites, and Territorial Defence
Authors: Day Richard T. , Elwood Robert W.
Published: March 2003

Tamarin monkeys, of the genus Saguinus, spend over half their lives at arboreal sleeping sites. The decision as to which site to use is likely to have considerable fitness consequences. These decisions about sleeping sites by three troops of golden‐handed tamarin Saguinus midas midas were examined over a 9‐mo period at a rainforest site in French Guiana. Data are presented on the physical nature of sleeping sites, their number, position within home ranges, and pattern of use and reuse, aspects of behaviour at retirement and egress, and predation attempts on the study troops. Cumulative plot analysis indicated that a tamarin troop used 30–40 sleeping sites in a 100‐day period, approximately half of which were used very infrequently, so that consecutive reuse was never greater than three nights. Sleeping trees were superior in architectural parameters and liana weight to non‐sleeping trees. There were no more sleeping sites than expected within the home range boundary region of the tamarins or in areas of overlap with the home ranges of neighbouring troops. Tamarins selected sleeping sites nearest to the last feeding site of the day on 25% of occasions. The study troops engaged in a number of activities that may reduce predation risk; raptor attacks on the study troops over 9 mo were frequent but unsuccessful. Tamarins often visited a sleeping site several hours before arrival, and were more likely to visit a site before use if they had not used it recently. The decision to select a sleeping site therefore involved knowledge of the previous frequency of use of that site.
Models of reproductive skew: A review and synthesis (Invited Article)
Authors: Johnstone Rufus A.
Published: March 2003

Animal societies vary markedly in reproductive skew, the extent to which breeding is monopolised by dominant individuals. In the last few years, a large number of different models have been developed to explain this variation. Here, I review existing models of reproductive skew, distinguishing between two basic types. Transactional models focus on group stability and the constraints this places on the division of reproduction. Compromise models, by contrast, ignore issues of group stability and view the division of reproduction as the outcome of a conflict in which each group member has a limited or partial ability to enforce its own optimum. I go on to show, however, that the division between transactional and compromise models is somewhat artificial, and that both approaches may be combined in a single, synthetic treatment. Different models of reproductive skew are thus better seen as special cases of a general underlying theory, rather than alternative paradigms. I conclude with a brief discussion of the possibilities and problems of empirically testing this unified theory of skew, and the prospects for future theoretical advances.
Multiple Copulations and Post‐Copulatory Guarding in a Free‐Living Population of Sika Deer (Cervus nippon)
Authors: Endo Akira , Doi Teruo
Published: March 2003

Multiple copulations by females and post‐copulatory guarding by males, were studied in a free‐living sika deer population, on Nozaki, an island of the Goto Group, off the coast of Kyushu, Japan. In 1990, 1991, and 1993, observations were carried out mainly on open grassland in the central section of the island. It was found, that 10 of 22 females in estrus copulated more than once and five of them copulated with several males (multi‐male copulations), while the remainder copulated with a single male (repeated copulation). Almost all copulations were followed by guarding behavior. Dominant males (DMs) guarded significantly longer and more effectively than subordinate males (SMs). Guarding was interrupted in four of the SMs, when the guarding male was forcibly driven away from a female by higher ranked males (‘take‐over’) and in three cases when the female left the guarding male spontaneously (‘escape’). The only interruption of guarding of a DM was caused when a lower ranked male sneaked towards the guarded female and mated with her briefly (sneak). Interruptions of guarding initiated by females (escapes) occurred more when they were guarded by SMs than by DMs.

Our results suggest that female sika deer indulge in multiple copulations to seek better genetic quality, rather than insuring fertility, enhancing genetic diversity, or avoiding harassment. The post‐copulatory guarding by DMs appears to be more effective in the prevention of additional copulations by females with other males than guarding by SMs. Moreover, SMs decrease the duration of the pre‐copulatory phase to achieve copulation before having to give way to DMs.
Properties of reaction zones associated with decay from pruning wounds in plantation‐grown Eucalyptus nitens
Authors: Barry K. M. , Pearce R. B. , Mohammed C. M.
Published: March 2003

Summary Decay columns resulting from naturally infected pruning wounds in 5‐ to 8‐year‐old plantation‐grown Eucalyptus nitens in Tasmania are interfaced by a reaction zone in the sapwood. The reaction zone is blue‐purple in colour and occasionally associated with a white zone at the reaction zone/healthy sapwood interface. A log incubation experiment has shown that the reaction zone is particularly durable against decay. The reaction zone is significantly drier than healthy sapwood, with lower levels of potassium, and a lower pH than both sapwood and heartwood. The increased total phenols levels and abundant tyloses in the reaction zone may be particularly important in defence.
Persistence of a biocontrol strain of Phlebiopsis gigantea in conifer stumps and its effects on within‐species genetic diversity
Authors: Vainio E. J. , Lipponen K. , Hantula J.
Published: March 2003

Fungal isolations and genetic fingerprinting were used to determine whether Phlebiopsis gigantea stump treatment against Heterobasidion annosum sl. using a single genotype (Rotstop) would affect the genetic diversity of P. gigantea populations. The survival time of P. gigantea was longer in Norway spruce (Picea abies) stumps compared to Scots pine (Pinus sylvestris) as no isolates were obtained from pine stumps 6 years after treatment, whereas in about half of the spruce stumps the fungus was still present. The usage of Rotstop did not seem to increase the occurrence of the fungus 5 years after the treatment in fresh (1‐year‐old) untreated stumps within the same forest stands. All the isolates from the 6‐year‐old treated spruce stumps were identical in genotype with the Rotstop‐strain, whereas all isolates from the fresh untreated spruce and pine stumps differed from it. Within the treated pine stand, the biocontrol usage seemed to have caused a slight reduction in genetic markers not related to Rotstop, but there were no statistically significant differences between the marker frequencies and the local natural population. Thus, Rotstop is not likely to cause any immediate threat to the genetic diversity of P. gigantea.
Molecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reaction
Authors: Winton L. M. , Hansen E. M.
Published: March 2003

A polymerase chain reaction (PCR)‐based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base‐pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water.
Viability of holm and cork oak seedlings from acorns sown in soils naturally infected with Phytophthora cinnamomi
Authors: Rodríguez‐Molina M. C. , Torres‐Vila L. M. , Blanco‐Santos A. , Núñez E. J. Palo , Torres‐Álvarez E.
Published: March 2003

The emergence and survival of pregerminated holm oak (Quercus ilex) and cork oak (Quercus suber) acorns from two ecologically different dehesas (Mediterranean open woodlands) were studied in two soils from these stands naturally infected with Phytophthora cinnamomi, and in the same soils previously sterilized in the autoclave. Phytophthora cinnamomi was consistently isolated from the radicles of all unemerged and all emerged but dead seedlings from the unsterilized substrates. Seedlings of holm oak were more susceptible to P. cinnamomi than those of cork oak. Mortality of holm oak seedlings was significantly different depending only on soil treatment (sterilized or unsterilized), and it was 100% in unsterilized soils, independent of acorn provenance and soil origin. Mortality of cork oak seedlings was significantly different depending on the acorn origin and soil treatment, and on the interactions acorn origin × soil origin and soil origin × soil treatment. The demonstrated high susceptibility of holm and cork oak young seedlings to P. cinnamomi could be a limiting factor in Mediterranean open woodlands (dehesas) not only in natural regeneration processes but also when reforestation by direct sowing is implemented.
Job satisfaction amongst nurses in an interim secure forensic unit in Wales
Authors: Burnard Philip , Morrison Paul , Phillips Ceri
Published: March 2003

All grades of nursing staff in a recently established interim secure forensic unit completed the Index of Work Satisfaction (IWS; n = 40). High levels of job satisfaction were achieved on four of the IWS subscales: (i) professional status; (ii) interaction; (iii) doctor–nurse relationship; and (iv) autonomy. Moderate levels of satisfaction were found on two of the subscales, task requirements and administration, while the salary subscale was the major area of dissatisfaction across the group. The high level of overall satisfaction in this area of psychiatric nursing may be regarded as a significant achievement in the development of new clinical services in what is regarded as a stressful area of nursing. Measures of job satisfaction may be useful benchmarks for evaluating future changes and developments in the service and for monitoring and improving the clinical work environment.
Scoping practice issues in the Australian mental health nursing workforce
Authors: Clinton Michael , Hazelton Mike
Published: March 2003

ABSTRACT

This is the third of four articles on the scoping study of the Australian mental health nursing workforce conducted on behalf of the Australian and New Zealand College of Mental Health Nurses (ANZCMHN) for the Australian Health Ministers Advisory Council (AHMAC) National Working Group on Mental Health (NWGMH). Its purpose is to focus on factors that significantly affect mental health nursing practice. The issues of advanced practice, regulation of nursing, accreditation, credentialling and demarcation with other disciplines are addressed.
THE SUCCESS OF MEN’S HEALTH NIGHTS AND HEALTH SESSIONS
Authors: Verrinder Adrian , Denner Bernard J.
Published: March 2003

ABSTRACT

Men’s health nights and men’s health sessions have proven to be remarkably successful in rural and some suburban regions of Victoria. In the rural regions, enough interest has been generated to run follow‐up health sessions on topics selected at the initial nights. Approximately 2000 men attended these events and 575 filled in questionnaires giving information such as age, occupation, health concerns and perceptions of health professionals. The results indicated that men’s health nights appeal to older men who are more likely to be professional or retired. These men saw cardiovascular disease, cancer and stress management as their main health concerns. The majority indicated that they would be interested in attending more men’s health sessions. The follow‐up sessions provide initial pathways by which men may address the issues of their own health. The data collected contributed to the development of the Men’s Awareness Network model for men’s health.