bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR Studies of the Action of Hypochlorous Acid on Native Pig Articular Cartilage
Authors: Schiller Jürgen , Arnhold Jürgen , Arnold Klaus
Published: September 2004

The action of sodium hypochlorite on pig articular cartilage was studied by 1H‐NMR spectroscopy to model some aspects of degradation processes of cartilage during rheumatoid arthritis. Two effects of NaOCl on cartilage polysaccharides have been observed. Hypochlorous acid causes an enhanced release of oligomeric polysaccharides from cartilage. The second effect concerns the degradation of N‐acetyl side chains of carbohydrates to acetate via a chlorinated transient product.

Signal intensities for N‐acetyl groups (≈2.0 ppm) increase during the first 2 h of incubation of cartilage with NaOCl. Then they decrease again. However, acetate (1.90 ppm) as the final product of degradation of N‐acetyl side chains increases continuously over the period of incubation with NaOCl.

In addition to polysaccharides, effects of NaOCl were only observed in cartilage samples on amino acids like alanine. The alanine resonance disappeared already at NaOCl concentrations where only small effects on cartilage polysaccharides have been observed.
Functional and Structural Changes of the Photosystem II Complex Induced by High Irradiance in Cyanobacterial Cells
Authors: Komenda Josef , Masojídek Jiří
Published: September 2004

A gradual disintegration of the photosystem II (PSII) complex, initiated by a release of the chlorophyll‐protein CP43, was identified during low‐temperature illumination of Synechococcus cells. This process was slower compared to the decline of the PSII primary charge separation activity, and much slower than the photoinactivation of oxygen evolution. All three processes were slowed down in the presence of diuron. The results indicate that when the PSII repair was blocked, the inactivation of charge separation activity and the release of CP43 preceded the degradation of the D1 protein. In contrast, a much faster degradation of D1 connected to its rapid exchange was triggered by inactivation of oxygen evolution, and no disassembly of PSII was needed. We propose the existence of two different mechanisms of D1 degradation in the cells of the thermophilic cyanobacterium Synechococcus elongatus.
Complete Amino Acid Sequence of a Zinc Metalloendoprotease from Streptomyces caespitosus
Authors: Harada Shigeharu , Kinoshita Takayoshi , Kasai Nobutami , Tsunasawa Susumu , Sakiyama Fumio
Published: September 2004

We determined the complete amino acid sequence of a zinc metalloendoprotease from Streptomyces caespitosus (ScNP). Peptide fragments obtained by digestion of Rcm‐ScNP with trypsin, ScNP and endoproteinase Asp‐N were purified by reverse‐phase HPLC and their amino acids were analyzed using an automatic sequencer. ScNP consisted of a single polypeptide chain of 132 amino acid residues with one disulfide bond between residues 99 and 112 (M, 14376). Thus, the number of amino acid residues determined for this enzyme is much lower than the number of residues previously reported for metalloendoproteases. The amino acid sequence indicated that although ScNP has the zinc‐binding motif, His‐Glu‐Xaa‐Xaa‐His, which is found at the active site of most zinc metalloendoproteases, it does not share overall significant similarity to the sequences of other zinc metalloendoproteases. Moreover, an analysis of the X‐ray structure of ScNP at 0.2–nm resolution (Kirisu et al., unpublished results) revealed that Asp93, together with two histidine residues in the zinc‐binding motif (His83 and His87) and a water molecule, is a zinc ligand. We propose that ScNP, which bears the HEXXHXXGXXD motif, represents a novel subfamily of zinc‐containing metalloendoproteases.
The Calmodulin‐binding Domain of the Inducible (Macrophage) Nitric Oxide Synthase
Authors: Anagli John , Hofmann Francesco , Quadroni Manfredo , Vorherr Thomas , Carafoli Ernesto
Published: September 2004

A domain in the inducible, macrophage nitric oxide (NO) synthase has been selected as the putative calmodulin‐binding site. The domain was synthesized as a peptide of 29 residues [P29, NO synthase‐(504–532)‐peptide], having the accepted hydrophobic/basic composition of calmodulin‐binding domains and containing, like most of them, an aromatic amino acid at its N‐terminus and a long chain aliphatic residue 12 amino acids downstream of it. A 34‐residue peptide from the synthase sequence [P34, NO synthase‐(499–532)‐peptide], consisting of peptide P29 and of the five extra N‐terminal amino acids, three of them basic, was also synthesized. Both peptides bound calmodulin in the presence as well as in the absence of Ca2+ (i.e. in the presence of excess EGTA). The KD of the binding in the presence of Ca2+ was ≤ nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin‐target enzyme, the Ca2+ pump of the plasma membrane.
Biochemical and Functional Properties of Photosystem II in Agranal Membranes from Maize Mesophyll and Bundle Sheath Chloroplasts
Authors: Bassi Roberto , Marquardt Jurgen , Lavergne Jérôme
Published: September 2004

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non‐denaturing lauryl β‐d‐iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light‐harvesting complex II (LHCII) that was somewhat over‐represented, while the minor antenna complexes CP26 and CP29 were under‐represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33‐kDa protein of the oxygen‐evolving enhancer polypeptides was not detectable nor was the ferredoxin‐NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3–(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect on the QA reoxidation. An additional specificity of these centers is the slow rate (50‐ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1–2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow β‐phase of the induction kinetics.
Purification of Recombinant GluR‐D Glutamate Receptor Produced in Sf21 Insect Cells
Authors: Kuusinen Arja , Arvola Milla , Oker‐Blom Christian , Keinänen Kari
Published: September 2004

GluR‐D glutamate receptors carrying FLAG and polyHis affinity tags at the N‐terminus and C‐terminus, respectively, were expressed in recombinant baculovirus‐infected Spodoptera frugiperda Sf21 cells. Affinity‐tagged receptors displayed ligand‐binding affinity (Kd=40 nM) and an expression level (Bmax 10–30 pmol/mg protein) similar to that of insect‐cell‐expressed wild‐type GluR‐D, as determined by [3H]‐α‐amino‐5‐hydroxy‐3‐methyl‐4‐isoxazole propionic acid ([3H]AMPA) binding. The receptor was solubilized in Triton X‐100, and purified using a two‐step protocol consisting of immobilized metal‐chelation affinity chromatography followed by immunoaffinity chromatography. The purified receptor preparation contained over 2000 pmol high‐affinity [3H]AMPA‐binding sites/mg protein, and migrated as a single 110‐kDa species in SDS/PAGE. Peptide: N‐glycosidase F treatment reduced the size of GluR‐D from 110 kDa to 100 kDa, indicating the presence of N‐linked glycans. Up to 100 μg purified GluR‐D was obtained from 11 Sf21 suspension culture (2–3×106 cells/ml). High‐level expression of affinity‐tagged GluRs in insect cells should be an efficient strategy to produce GluR subtypes for biochemical and structural studies.
Biochemical Characterization of the 8‐hydroxy‐5‐deazaflavin‐reactive Hydrogenase from Methanosarcina barkeri Fusaro
Authors: Michel Regina , Massanz Christian , Kostka Susanne , Richter Monika , Fiebig Klaus
Published: September 2004

The membrane‐associated coenzyme F420‐reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95‐fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420‐reducing activity (82.8 μmol · min−1· mg protein−1 versus 11.5 μmol · min−1· mg protein−1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79–88]. The predominant coenzyme F420‐reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non‐identical subunits with apparent molecular masses of 48 (α), 33 (β), and 30 kDa (γ), apparently in a stoichiometry of α2β2γ1. This minimal coenzyme F420‐reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198‐kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28–32 mol non‐heme iron, and 34 mol acid‐labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N‐termini of the putative nickel‐binding subunits of most [NiFe]‐ and [NiFe]Sehydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420‐reducing activity was obtained with reductively reactivated enzyme at 55°C in the pH range 6.5–7.25. The Km, values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3μM and 4μM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5×107M−1· s−1 with coenzyme F420 and 6.9×107 M−1· S−1 with methylviologen.
Characterisation of a Second, Apparently Inactive, Copy of the Bovine β‐lactoglobulin Gene
Authors: Passey Robert J. , Mackinlay Antony G.
Published: September 2004

A bovine cosmid clone was isolated which contains the previously characterised β‐lactoglobulin gene and, in addition, a related sequence which appears to be a β‐lactoglobulin pseudogene. The total length of the pseudogene, as determined by DNA sequencing, is 4.8 kb, similar to that of β‐lactoglobulin. Both genes are in the same orientation and are separated by approximately 14 kb intergenic sequence. Although most of introns I‐V are extremely divergent, the exon sequences are clearly related, exons I‐V exhibiting nucleotide sequence similarities in the range 60–87%. Exons VI and VII, together with the final intron, comprise a region of sequence extending over 730 bp, which displays 92.5% identity to the corresponding β‐lactoglobulin sequence. It is suggested that this is the result of a recent gene conversion event involving conversion of the pseudogene by the authentic β‐lactoglobulin gene. Identification of the new lactoglobulin sequence as a pseudogene is based on the occurrence of a stop codon in exon V. Comparison of the inferred translation product encoded by the pseudogene before its mutational inactivation, with the sequences of equine and feline β‐lactoglobulins I and II, indicates that the bovine pseudogene is more closely related to these type‐II lactoglobulin sequences than to the type‐I sequences.
A Precursor‐product Relationship in Molluscan Sperm Proteins from Ensis minor
Authors: Bandiera Antonella , Patel Umesh A. , Manfioletti Guidalberto , Rustighi Alessandra , Giancotti Vincenzo , Crane‐Robinson Colyn
Published: September 2004

A cDNA library prepared from mRNA extracted from immature male gonads of the bivalve mollusc Ensis minor (razor shell) was probed with a 133‐bp reverse‐transcriptase PCR product corresponding to a segment of the sperm protein EM6 [Giancotti, V., Russo, E., Gasparini, M., Serrano, D., Del Piero, D., Thorne, A. W., Cary, P. D. & Crane‐Robinson, C. (1993) Eur. J. Biochem. 136, 509–516]. A single 1.5‐kb clone was found to encode both sperm proteins EM1 and EM6. Mass spectrometry was used to define the C‐terminus of EM1, and since the N‐terminus of EM6 is known from Edman degradation, this showed that the pentapeptide NTNNS must be lost on proteolytic processing. Both EM1 and EM6 contain highly repeated amino acid sequences, suggestive of extended structures. EM1 contains seven tandem repeats of the dipeptide S(K/R), followed by six potential cdc2 phosphorylation sites and seven repeats of the octapeptide KRSASKKR, with occasional K/R substitutions. EM6 contains a globular domain preceded by 17 almost identical uninterupted tandem repeats of the motif KKRSXSRKRSAS, where X is charged. Its C‐terminus contains 15 short basic clusters. Assignment of EM1 and EM6 to the established categories of molluscan sperm proteins [PLI, PLII, PLIII, PLIV: Ausio, J. (1992) Mol. Cell. Biochem. 115, 163–172] is discussed.
His103 in Yeast Transketolase is Required for Substrate Recognition and Catalysis
Authors: Wikner Christer , Meshalkina Ludmilla , Nilsson Ulrika , Bäckström Stefan , Lindqvist Ylva , Schneider Gunter
Published: September 2004

Crystallographic studies of thiamin‐diphosphate‐dependent transketolase from Saccharomyces cerevisiae suggested the invariant active‐site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site‐directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side‐chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km, values for the acceptor substrate ribose 5‐phosphate were similar to the Km value for wild‐type transketolase. However, the Km, value for the donor substrate, xylulose 5‐phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates.
Expression of Functional Ricin B Chain using the Baculovirus System
Authors: Ferrini Jean‐Bernard , Martin Marianne , Taupiac Marie‐Pierre , Beaumelle Bruno
Published: September 2004

The ricin B chain (RTB) was expressed using a baculovirus expression system. The RTB coding sequence downstream of the preproricin signal sequence was inserted in the baculovirus transfer vector pM34T. After cotransfection of Spodoptera frugiperda Sf9 cells with linearized baculovirus DNA, recombinant viruses were selected. cloned and amplified. Upon infection of Sf9 cells with these recombinant baculoviruses, RTB production was revealed by immunoblotting. RTB expression using this system was optimum 72 h after infection of the cells at a multiplicity of infection of 3. RTB produced was glycosylated and had an apparent molecular mass of 34 kDa. Most of the signal sequence was removed, but the resulting recombinant RTB had a 13‐residue N‐terminus extension. Immunofluorescence analysis showed that this protein was located in the endoplasmic reticulum/Golgi region of the cell. RTB was not present at the plasma membrane. Secretion was enhanced by the addition of lactose to the cell‐culture medium up to 50 mM. Purification was achieved from both cells and media using immobilized lactose and the lectin activity of RTB. Results obtained with the purified recombinant protein (more than 2 mg/l culture) were identical to those obtained with native RTB in all assays for biological activity; binding, internaliza‐tion and reassociation with the ricin A chain to produce toxic ricin. Moreover, the RTB translocation capacity was not altered by the N‐terminal peptide, showing that recombinant RTB could be used to deliver antigenic peptides to the cytosol for the induction of cell‐mediated immunity.
Cordycepin Blocks Recovery of Non‐Heat‐Shock mRNA Translation following Heat Shock in Drosophila
Authors: Duncan Roger F.
Published: September 2004

Treatment of cells with cordycepin (3‐deoxyadenosine), an inhibitor of cytoplasmic adenylation, blocks the restoration of normal translation following heat shock. Cordycepin also reduces heat‐shock protein 70 (Hsp70) protein synthesis greater than 10‐fold, while having little to no effect on mRNA accumulation. Parallel analysis of the poly(A)‐binding protein detects no change in its abundance during heat shock or subsequent recovery. These results suggest that normal, non‐heat‐shock mRNA translational repression during heat shock may be caused by deadenylation, and that readenylation is required for restoration of activity. However, three independent analyses of the adenylation status of mRNAs during heat shock and recovery indicate that no significant changes in polyadenylation occur. (a) The total poly (A) content decreases by only about 10% during heat shock; (b) the size of the poly (A) tract decreases only marginally, from an average length of 75–90 nucleotides in non‐heated cells to 45–60 nucleotides during heat shock; (c) virtually all mRNAs bind to oligo d(T)‐cellulose, whether extracted from normal‐temperature. heat‐shock or recovered cells. Our results are most consistent with a model where the process of readenylation, rather than the specific poly(A) tail length, influences translational activation during recovery, paralleling a proposed model for the activation of translation during Xenopus oocyte maturation.
A CCACC Motif Mediates Negative Transcriptional Regulation of the Human Erythropoietin Receptor
Authors: Maouche Leïla , Lucien Nicole , Cartron Jean Pierre , Chrétien Stany
Published: September 2004

We have previously shown that the +79 to +135 fragment of the human erythropoietin receptor (Epo‐R) acts negatively on the transcriptional activity and confers erythroid specificity to the gene [Maouche, L., Cartron, J.‐P. & Chrétien, S. (1994) Nucleic Acids Res. 22, 338–346]. In this work, we demonstrate that this effect is mediated by a CCACC motif that binds weakly to the simian virus 40 protein 1 (Sp1) factor and that the increase of the affinity for Sp1 augments transcription inhibition. The repression is not restricted to the human Epo‐R promoter, although it seems more efficient on heterologous promoters of erythroid genes.

In chloramphenicol acetyl transferase constructs containing the mouse Epo‐R promoter, rearranged by retroviral long terminal repeat (LTR) insertion of murine erythroleukemia cell lines, we found that positioning the CCACC motif 3′ to the LTR represses the transcriptional activity mediated by the LTR in non‐erythroid cells.

These results demonstrate that Epo‐R gene expression is negatively regulated by a CCACC or a GC box‐binding factor, which is most likely identical to the Sp1 transcription protein. Further data suggest that Sp1‐mediated negative regulation is not the result of a direct competition between Sp1 and another DNA‐binding protein.
The Phosphoglycerate Kinase and glsyceraldehyde‐3‐phosphate Dehydrogenase Genes from the Thermophilic Archaeon Sulfolobus Solfataricus Overlap by 8‐bp
Authors: Jones Carol E. , Fleming Toni M. , Cowan Don A. , Littlechild Jennifer A. , Piper Peter W.
Published: September 2004

The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde‐3‐phosphate dehydrogenase (GraP‐DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced. PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene. This was then used to screen a genomic library to isolate the full length pgk gene. A 2.5‐kb Bg/II fragment of S. solfataricus DNA contained both the pgk gene and the gap gene immediately downstream. Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evidence that the two genes are co‐transcribed was obtained by Northern‐blot analysis. The S. solfataricus PGK amino acid sequence shows 43 % and 45 % identity to the PGK sequences of the Archaea Methano‐bacterium bryantii and Methanothermus fervidus, respectively. High level expression of the S. solfataricus PGK and GraP‐DH in Escherichia coli was achieved, with heat treatment at 80°C proving an effective first step in the purification of these recombinant enzymes from extracts of the E. coli host. Purified recombinant S. solfutaricus PGK and GraP‐DH showed half lives of 39 min and 17 h, respectively, at 80°C. Unlike bacterial GraP‐DH enzymes, S. solfaturicus GraP‐DH was able to use both NAD+ and NADP+ as cofactors, but exhibited a marked preference for NADP+
Absolute Requirement of Ammonium Sulfate for Reconstitution of Active 70s Ribosomes from the Extreme Halophilic Archaeon Haloferax Mediterranei
Authors: Sánchez Emma , Amils Ricardo
Published: September 2004

Active 70s ribosomes from the halophilic archaeon Haloferax mediterranei have been reconstituted from their isolated rRNAs and proteins. The reconstitution procedure consists of a two‐step incubation; first with 1 M ammonium sulfate and 100 mM magnesium acetate for 1 h at 42°C, followed by a 90‐min incubation at 50°C after increasing the ammonium sulfate to 2 M final concentration. The total reconstitution of halophilic 70S ribosomes is a process with its own identity, which does not correspond to the conditions required for the reconstitution of the isolated subunits. Ammonium sulfate is the only salt capable of promoting the assembly of active ribosomes. The increase of ammonium sulfate salts in the second incubation step is obligatory for the isolation of functional particles.
Purification of Active E1α2β2 of Pseudomonas Putida Branched‐Chain‐Oxoacid Dehydrogenase
Authors: Hester Kathryn , Luo Jinhe , Burns Gayle , Braswell Emory H. , Sokatch John R.
Published: September 2004

Active E1 component of Pseudomonas putida branched‐chain‐oxoacid dehydrogenase was purified from P. putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdA1 and bkdA2 (encoding the α and β) subunits). Expression was inducible, however, 45– 39– and 37‐kDa proteins were produced instead of the expected 45‐kDa and 37‐kDa proteins. The 45‐kDa protein was identified as E1α and the 37‐kDa and 39‐kDa proteins were identified as separate translational products of bkdA2 by their N‐terminal sequences. The N‐terminal amino acid of the 39‐kDa protein was leucine instead of methionine. The 45–, 39– and 37‐kDa proteins were also produced in wild‐type P. putida. Translation of bkdA1 and bkdA2 from an Escherichia coli expression plasmid produced only 45‐kDa and 39‐kDa proteins, with N‐terminal methionine on the 39‐kDa protein. The insertion of guanine residues 5′ to the first ATG of bkdA2 did not affect expression of E1β in P. putida including the N‐terminal leucine which appears to eliminate the possibility of ribosome jumping. The Z‐average molecular mass of the El component was determined by sedimentation equilibrium to be 172±9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm. E1α Ser313, which is homologous to the phosphorylated residue of rat liver E1α, was converted to alanine resulting in about a twofold increase in Km, but no change in Kcat. S315A and S319A mutations had no effect on Km or Kcat, indicating that these residues do not play a major part in catalysis of E1α2β2.
Folding of the Squash Trypsin Inhibitor EETI II
Authors: Heitz Annie , Chiche Laurent , Le‐Nguyen Dung , Castro Bertrand
Published: September 2004

A peptide, corresponding to the entire sequence of the squash trypsin inhibitor EETI II(Ecballium elateriumtrypsin inhibitor) in which the six cysteines, engaged in three disulphide bridges in native EETI II, have been replaced by six serines, has been synthesised. CD, Fourier‐transform infrared spectroscopy (FTIR) and1H‐NMR studies of this peptide revealed that some secondary structures present in native EETI II are still populated in the absence of disulphide bonds. Native‐like secondary structures were observed for segments 10–15 (helix), 16–19 and 22–25 (reverse turns) but no native tertiary interaction was detected. However, a non‐native local interaction between the aromatic ring of Phe26 and the amide group of Gly28 was observed. It is hypothesised that the 10–15, 16–19 and 22–25 native‐like local conformations could play a major role in the early folding of EETI II.
Carotene Desaturation is Linked to a Respiratory Redox Pathway in Narcissus Pseudonarcissus Chromoplast Membranes
Authors: Nievelstein Vitus , Vandekerckhove Joel , Tadros Monier H. , Lintig Joharmes V. , Nitschke Wolfgang , Beyer Peter
Published: September 2004

The enzymic activity of phytoene desaturase in Nurcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox‐active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone‐rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox‐active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43‐kDa NAD(P)H: quinone oxidoreductase, which is organized as a homodimer (23±3 kDasubunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23‐kDa protein of the oxygen‐evolving complex of photosystem II, but no information about the N‐terminal organization of the oxidoreductase could be obtained. During flower development, the steady‐state concentration of the corresponding mRNA is up‐regulated, indicating a specific function of the gene product in chlorophyll‐free chromoplasts.
Affinity Labeling of Recombinant Ricin A Chain with Procion Blue MX‐R
Authors: Alderton Wendy K. , Thatcher David , Lowe Christopher R.
Published: September 2004

Recombinant ricin A chain was irreversibly modified by Procion blue MX‐R, a dichlorotriazinyl analogue of Cibacron blue F3G‐A, at pH 7.5 and 4°C in 90 h with over 95% loss of activity in an in vitro translation assay. The presence of total yeast RNA reduced the covalent attachment of Procion blue MX‐R to ricin A chain. Quantitatively modified ricin A chain contained 2 mol Procion blue MX‐R/mol 29‐kDa subunit. Tryptic digestion and resolution of the peptides by reverse‐phase high‐performance liquid chromatography yields a blue peptide corresponding to Gln5 – Arg26 of ricin A chain. Thus, a likely dye‐binding site on recombinant ricin A was identified. This region is removed from the active‐site cleft of recombinant ricin A but may be involved in its substrate binding.
2‐Chloro‐2′ ‐deoxyadenosine (Cladribine) and its Analogues are Good Substrates and Potent Selective Inhibitors of Escherichia coli Purine‐Nucleoside Phosphorylase
Authors: Bzowska Agnieszka , Kazimierczuk Zygmunt
Published: September 2004

2‐Chloro‐2′‐deoxyadenosine (CldAdo), a nucleoside that has proven useful in the treatment of several chronic lymphoid malignancies, and its analogue, 2‐bromo‐2′‐deoxyadenosine, are both effective inhibitors of the bacterial (Escherichia coli) purine‐nucleoside phosphorylase (PNP), with Ki values of 4.5 μM and 6.3 μM, respectively. The examination of a series of base‐modified analogues of CldAdo has shown that several other compounds have similar inhibitor properties, and has indicated that 6‐benzyloxy‐2‐chloro‐9–(2′‐deoxy‐β‐d‐ribofuranosyl)purine is the most potent inhibitor with a Ki, value of 0.5 μM, competitive with respect to inosine (Ino).

CldAdo itself and its base‐modified analogues, discounting those substituted at C(8), are also substrates for the E. coli PNP and undergo rapid glycosidic bond cleavage. CldAdo is degraded with substrate efficiency, i.e. Vmax/Km similar to that observed for Ino (130%), although the individual kinetic constants, Km and Vmax are both approximately an order of magnitude lower than for Ino.

All compounds tested are totally inactive as substrates and inhibitors for mammalian (calf spleen) PNP and therefore constitute a new class of potent selective, although cleavable, inhibitors of bacterial phosphorylases.

8‐Bromo‐2‐chloro‐2′‐deoxyadenosine and 8‐thio‐2‐chloro‐2′‐deoxyadenosine are the only base‐modified CldAdo derivatives showing inhibitory activity against MOLT‐3 (acute T‐cell leukemia) and U‐937 (histiocytic lymphoma) cells and, as shown in this study, are resistant to degradation by E. coli PNP. The above‐mentioned results suggest that both analogues could be effective as oral cytotoxic agents that are noncleavable by enteric bacteria.
Effect of pH on Sheep Liver Sorbitol Dehydrogenase Steady‐State Kinetics
Authors: Lindstad Rune I. , Mckinley‐Mckee John S.
Published: September 2004

The variation with pH of the kinetic parameters for sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase has been studied over the pH 5–10 range. The reaction is compulsory ordered in both directions with the coenzyme as the leading substrate, and the rate‐determining step in either direction is the enzyme‐coenzyme product dissociation. Throughout the pH range, the lack of a primary kinetic isotope effect on Vm with (2H8) sorbitol confirms that the ternary complexes are not of rate‐determining significance under maximum velocity conditions.

The association rate constants for NAD and NADH increase and decrease, respectively, towards high pH. NAD binding to the enzyme is dependent upon pK values of 9.2 and 9.6. Whereas the dissociation rate constant for NAD release from the enzyme shows no pronounced variation with pH, NADH release is dependent upon pK values of 7.2 and 7.7.

The kinetic constants that characterize the dependence on substrate concentration of the steady‐state rate of catalysis vary with pH in accordance with a single pK of 7.1 for sorbitol oxidation and of 7.7 for fructose reduction. These pK values reflect the ionization properties of a catalytically essential group, which is tentatively considered to be either the H2O/OH− ligand binding to the catalytic zinc atom or a histidine residue. Catalysis by sorbitol dehydrogenase, due to the absence of a second ionization contribution, appears not to involve any obligatory step of proton transfer to solution at the ternary complex level. A mechanism for sorbitol dehydrogenase catalysis is proposed.
Structural Studies of the Putative O‐Specific Polysaccharide of Acinetobacter baumannii O2 containing 3,6‐dideoxy‐3–N ‐(d‐3‐hydroxybutyryl)amino‐d‐galactose
Authors: Haseley Simon R. , Wilkinson Stephen C.
Published: September 2004

A polysaccharide containing d‐galactose, 2‐deoxy‐2–N ‐acetylamino‐d‐galactose and 3,6‐dideoxy‐3–N ‐(d‐3‐hydroxybutyryl)amino‐d‐galactose, probably corresponding to the lipopolysaccharide side chain, was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain O2. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched hexasaccharide of the structure shown, where Fuc3N represents 3‐amino‐3,6‐dideoxygalac‐tose and R represents d‐3‐hydroxybutyryl. Serological tests indicated that the polymer corresponded to the O2 antigen.
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A Glycoprotein Inhibitor of Pectin Methylesterase in Kiwi Fruit
Authors: Giovane Alfonso , Balestrieri Ciro , Quagliuolo Lucio , Castaldo Domenico , Servillo Luigi
Published: September 2004

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single‐step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel‐filtration experiments.

The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N‐glycosidase F after protein denaturation and, to a minor extent, by digestion with N‐glycosidase H. No glycidic residue could be removed by digesting the native protein with those N‐glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.
UDP Galactose: Ceramide Galactosyltransferase and Glutamate/Aspartate Transporter
Authors: Schulte Stephan , Stoffel Wilhelm
Published: September 2004

The oligodendrocyte‐specific UDP‐galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte‐ and myelin‐specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton‐X‐100‐solubilized rat brain micro‐somes by ion exchange, dye ligand and lectin affinity chromatography as a 64‐kDa protein homogenous in SDS/PAGE. It copurified with the brain‐specific Na+‐dependent high‐affinity l‐glutamate/aspartate neurotransmitter transporter (GLAST‐1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high‐mannose glycoprotein and GLAST‐1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70‐kDa isoform of GLAST‐1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70‐kDa isoform differs only in the degree of glycosylation from the 64‐kDa GLAST‐1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein‐chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST‐I were successfully applied to the cloning of CGT and the first high‐affinity glutamate neurotransmitter transporter (GLAST‐1) in glia of the central nervous system as well.
The influence of energy sources on the proteolysis of a recombinant staphylococcal protein A in Escherichia coli
Authors: Yang Shaojun , Enfors Sven‐Olof
Published: September 2004

The kinetics of proteolysis of a recombinant staphylococcal protein A in Escherichia coli were studied by a Western‐blotting‐based method. The proteolysis constants obtained from this method are very close to those obtained from the traditional radioactive pulse‐chase technique. Protein A was selectively degraded to a great extent, while host proteins were quite stable after heat induction of protein A expression. The proteolysis of protein A was much faster in the presence of energy sources compared to when cells were starved of energy. The degradation rate constants are 2.8 h−1 in the presence of 10 g/l glucose and about 0.4 h−1 in the absence of any external carbon source. The supplementation of glucose to the medium at 0–100 mg/l caused a gradual increase of proteolysis of protein A, but the proteolysis was saturated when the concentration of glucose exceeded 200 mg/l at a cell concentration of about 0.36 g/l. The respiration inhibitor sodium azide completely inhibited the degradation of protein A in glucose‐free salt medium but had almost no effect in the presence of glucose. Therefore, the proteolysis process is energy dependent but the energy supply rate obtained by fermentation of glucose is enough to meet this requirement. The proteolysis rate increased with the temperature in the interval 5–45°C but was then reduced due to damage of the proteolysis system by high temperature. At 60°C, the proteolysis ceased completely within 30 min.
Borderline pathology in children and adolescents
Authors: Meekings Charles , O’Brien Louise
Published: September 2004

ABSTRACT:   Mental health nurses have historically been pessimistic about and often unsym­pathetic towards clients diagnosed with borderline personality disorder. By the time these clients reach adult mental health services their behaviours are often difficult to manage and they often suffer significant re‐victimization by health services. Questions need to be raised about how best to avert the consolidation of the problems associated with the disorder. This paper explores the concept of ‘borderline pathology’ in children and adolescents and examines the best available evidence for utilizing an early identification and intervention model for children and adolescents who exhibit this constellation of symptoms.
Emotional intelligence in mental health nurses talking about practice
Authors: Akerjordet Kristin , Severinsson Elisabeth
Published: September 2004

ABSTRACT:  The aim of this study was to explore mental health nurses’ experiences of emotional intelligence (EI) in their nursing practice by means of qualitative interviews. The interview questions where developed from the literature on EI. This study used a hermeneutic analysis. Four main themes emerged: relationship with the patient; the substance of supervision; motivation; and responsibility. It was concluded that EI stimulates the search for a deeper understanding of a professional mental health nursing identity. Emotional learning and maturation processes are central to professional competence, that is, personal growth and development. In addition, the moral character of the mental health nurse in relation to clinical practice is of importance. The findings imply multiple types of intelligence related to nursing science as well as further research possibilities within the area of EI.
An evaluation of an educational program for clinical supervision
Authors: Hancox Kerrie , Lynch Lisa , Happell Brenda , Biondo Sebastiana
Published: September 2004

ABSTRACT:  Clinical supervision for mental health nurses has become an area of priority since the implementation of the Enterprise Bargaining Agreement of August 2000 in Victoria. Clinical supervision has been identified as a strategy to improve both the job satisfaction of nurses and the quality of care provided to consumers. A review of the literature suggests that adequate education for supervisors is crucial if this strategy is to prove successful. In response to this identified need, the Centre for Psychiatric Nursing Research and Practice developed the ‘Clinical Supervision for Health Professionals’ program. The evaluation of participants from the first 2 years of the program (n = 63) are mentioned here. The findings suggest a high level of satisfaction with the education provided and that it has been successful in improving participants’ attitudes towards, and level of confidence in providing, clinical supervision.
Apportioning our time and energy: oral presentation, poster, journal article or other?
Authors: Cleary Michelle , Walter Garry
Published: September 2004

ABSTRACT:  There is a general expectation for health service employees to present their work in oral or written format to showcase clinical ideas, innovations, service developments, and quality and research initiatives. This research note outlines the types of forums where work can be presented and highlights their relative merits. It is anticipated that this discussion will be of interest to clinicians, managers and researchers when considering where best to present their work.
Vertical Price Control and Parallel Imports: Theory and Evidence
Authors: Maskus Keith E. , Chen Yongmin
Published: September 2004

The paper analyzes parallel imports, or goods traded without the authorization of a trademark owner. Parallel imports have multiple causes, including vertical price control, which the authors model. A manufacturer selling its product through an independent agent sets the wholesale price sufficiently low to induce a desired retail price abroad. This permits the agent to sell the product profitably in the originating market. Combined social surplus decreases and then increases in the cost of parallel trade. Restricting parallel imports benefits the manufacturer, but could raise or reduce global surplus. The econometric analysis indicates that the vertical‐control explanation of parallel imports is important.
Entry, Expansion, and Intensity in the US Export Boom, 1987–1992
Authors: Bernard Andrew B. , Jensen J. Bradford
Published: September 2004

US exports grew at 10.3% per year from 1987 to 1992, far faster than the economy as a whole. This paper examines sources of the manufacturing export boom, including entry, firm expansion, and export intensity. Most of the increase in exports came from increasing export intensity at existing exporters rather than from new entry into exporting. The small role of entry relative to export intensity offers support for the importance of sunk costs in the export market. Changes in exchange rates and rises in foreign income drove most of the export increase, while plant productivity increases played a smaller role.
Adam Smith and Noneconomic Objectives
Authors: Elmslie Bruce
Published: September 2004

This note looks at Smith's critiques of bounties (read “subsidies”) on exports. Smith's analysis of bounties foreshadowed the basic trade policy framework of distortions and noneconomic objectives. While Smith did not develop the theory of distortions, he captured the logic of the theory of noneconomic objectives and actually ranked policy alternatives based upon their relative social costs. Corden (1957) first ranked tariffs versus production subsidies within a noneconomic objectives framework, and Johnson (1960) first used the term in its modern sense, but the formulation of the general framework lies with Smith.
Patterns of Corporate Financing and Financial System Convergence in Europe
Authors: Murinde Victor , Agung Juda , Mullineux Andy
Published: September 2004

The paper investigates the possibility of convergence in the European Union (EU) in terms of the patterns of corporate financing by banks, bond markets, and stock markets; and in the context of whether the economies are converging towards an Anglo‐Saxon (capital‐market‐oriented) or a continental (bank‐oriented) financial system. GMM estimation of a dynamic fixed‐effects model is implemented to test for conditional and unconditional convergence using a panel of flow of funds data for the period 1972–1996 for seven EU member countries. It is found that the pattern of corporate financing is consistent with the pecking order theory of financing choices. Overall, the evidence suggests convergence of the EU financial systems on a variant of the Anglo‐Saxon model, depicting heavy reliance on internal financing as well as direct financing via equity and bond markets, while bank debt is becoming relatively less important.
Tangible User Interfaces (TUIs): A Novel Paradigm for GIS
Authors: Ratti Carlo , Wang Yao , Ishii Hiroshi , Piper Ben , Frenchman Dennis
Published: September 2004

In recent years, an increasing amount of effort has gone into the design of GIS user interfaces. On the one hand, Graphical User Interfaces (GUIs) with a high degree of sophistication have replaced line‐driven commands of first‐generation GIS. On the other hand, a number of alternative approaches have been suggested, most notably those based on Virtual Environments (VEs). In this paper we discuss a novel interface for GIS, which springs from recent work carried out in the field of Tangible User Interfaces (TUIs). The philosophy behind TUIs is to allow people to interact with computers via familiar tangible objects, therefore taking advantage of the richness of the tactile world combined with the power of numerical simulations. Two experimental systems, named Illuminating Clay and SandScape, are described here and their applications to GIS are examined. Conclusions suggest that these interfaces might streamline the landscape design process and result in a more effective use of GIS, especially when distributed decision‐making and discussion with non‐experts are involved.
Partitioning Spatial Model Uncertainty Based on Joint Spatial Simulation
Authors: Gertner George Z , Fang Shoufan , Wang Guangxing , Anderson Alan
Published: September 2004

In this study, an uncertainty analysis procedure for joint sequential simulation of multiple attributes of spatially explicit models used in geographical informational systems was developed based on regression analysis. This procedure utilizes information obtained from joint sequential simulation to establish the relationship between model uncertainty and variation of model inputs. Using this procedure, model variance can be partitioned by model input parameters on a cell by cell basis. In the partitioning, the correlation of neighboring cells is accounted for. With traditional uncertainty analysis methods, this is not possible. In a case study, spatial variation of soil erodibility from a joint sequential simulation of soil properties was analyzed. The results showed that the regression approach is a very effective method in the analysis of the relationship between variation of the model output and model input parameters. It was also shown for the case study that: (1) the uncertainty of soil erodibility of a cell is mainly propagated from its own soil properties; (2) the interactions of soil properties of neighboring cells could reduce uncertainty of soil erodibility; (3) it is sufficient for uncertainty analysis to include the nearest three neighboring cell groups; and (4) the largest uncertainty contributors vary by soil properties and location.