bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of the structural requirements for DNA cleavage by the isoschizomers HaeIII, BspRI and BsuRI
Authors: WOLFES Heiner , FLIESS Anja , PINGOUD Alfred
Published: March 2005

We have investigated the structural requirements for DNA cleavage by the isoschizomers HaeIII, BspRI and BsuRI which recognize the sequence ‐d(GGCC)‐. For this purpose decadeoxynucleotides were synthesized by the solid‐phase phosphotriester method and purified by high‐performance liquid chromatography. The kinetics of cleavage of these oligodeoxynucleotides were determined for the three isoschizomers with the following results.

1. The sequence adjacent to the recognition site strongly influences the rate of cleavage. The preference is qualitatively the same for all three enzymes: AGGCCT > TGGCCA > GGGCCC ∼ CGGCCG, and follows the thermal stability of the different decanucleotides.

2. Substitutions within the recognition site, namely dI for dG and dU for dC, affect the rate of cleavage differently for the three enzymes. The results can be rationalized in terms of an interaction of HaeIII with major and minor groove of the DNA, of BspRI mainly with the minor groove and of BsuRI with the major groove of DNA. It is obvious from our data that the mechanism of recognition of the same site is different for the three isoschizomers.
Synthesis of Escherichia coli carbomoylphosphate synthetase initiates at a UUG codon
Authors: WEYENS Guy , ROSE Keith , FALMAGNE Paul , GLANSDORFF Nicolas , PIÉRARD André
Published: March 2005

The ribosome binding region of the messenger RNA for the Esherichia coli car A gene contains two adjacent putative translational start codons, UUG and AUU, both of them unusual. By Edman degradation and mass spectrometry of purified car A protein, we show that only UUG is used in vivo. Translation initiation at UUG in car A appears about half as efficient as at AUG in lacZ.
The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols
Authors: EGGELING Lothar , SAHM Hermann
Published: March 2005

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram‐positive bacteria Rhodococcus erythropolis.

1. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross‐linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase.

2. A heat‐stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofousing revealed that the cofactor is a weak acid with a pK of about 6.5.

3. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate.

It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.
Characterization of the high‐affinity interaction between human plasminogen and pro‐urokinase
Authors: LIJNEN H. Roger , ZAMARRON Concepcion , COLLEN Désiré
Published: March 2005

Activation of human Glu‐plasminogen, Lys‐plasminogen and low‐Mr plasminogen (lacking lysine‐binding sites) by pro‐urokinase (pro‐UK), obtained from a human lung adenocarcinoma cell line (Calu‐3, ATCC). obeys Michaelis‐Menten kinetics. Activation ocurs with a comparable affinity (Km 0.40–0.77 μM), while the catalytic rate constant (kcat) is comparable for Glu‐plasminogen (0.0022 s−1) and low‐Mr plasminogen (0.0034s−1), but is somewhat higher for Lys‐plasminogen (0.0106s−1). The rate of activation of plasminogen by pro‐UK is not significantly influenced by the presence of 6‐aminohexanoic acid, purified fragments LBS I or LBS II or histidinerich glycoprotein, indicating that the high affinity of pro‐UK for plasminogen is not mediated via the high‐affinity lysine‐binding site of plasminogen located in kringles 1–3 (LBS I) nor via the low‐affinity lysine‐binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high‐affinity interaction with pro‐UK thus appear to be located within the low‐Mr plasminogen moiety.
Amino acid sequence of a sea anemone toxin from Parasicyonis actinostoloides
Authors: NISHIDA Shigeru , FUJITA Shozo , WARASHINA Akira , SATAKE Mei , TAMIYA Nobuo
Published: March 2005

Amino‐acid sequence of a toxin from sea anemone, Parasicyonis actinostoloides, is determined. The toxin consists of 31 amino acid residues and is cross‐linked with four disulphide bridges. The sequence has some similarity to that of toxin III and no similarity to those of toxin I and toxin II both from sea anemone, Anemonia sulcata, or to that of Anthopleurin A from Anthopleura xanthogrammica.
Creatine phosphokinase: isoenzymes in Torpedo marmorata
Authors: WITZEMANN Veit
Published: March 2005

Creatine phosphokinase (ATP:creatine N‐phosphotransferase, EC 2.7.3.2) is the major constituent of the ‘low‐salt‐soluble’ proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43 000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti‐(electric organ creatine phosphokinase) antibodies are specific for the muscle‐type enzyme and do not cross‐react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine‐receptor‐enriched membranes show that this enzyme is as the ‘low‐salt‐soluble’ electric organ enzyme of the muscle‐specific type. In vitro translation of electric organ poly(A)‐rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain‐type polypeptides are synthesized. The subunits of the brain‐specific enzyme differ in molecular mass (Mr∼ 42 000) and isoelectric properties (pI ∼ 7.0–7.2). The unexpected finding that the brain forms are more basic than the muscle‐specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion‐exchange chromatography properties.
Characterization of the bovine lens plasma membrane substrates for cAMP‐dependent protein kinase
Authors: LOUIS Charles F. , JOHNSON Ross , JOHNSON Keith , TURNQUIST Janet
Published: March 2005

cAMP‐dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic‐reticulum‐bound protein phosphatase activity. The 26 kDa component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N‐chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.
A model for intracellular complexation between gene‐5 protein and bacteriophage fd DNA
Authors: BRAYER Gary D. , McPHERSON Alexander
Published: March 2005

A structural model for the helical intracellular complex formed between the gene‐5 DNA‐binding protein (G5BP; ∼ 1274 copies) and bacteriophage fd DNA has been derived by an atomic‐contact anaysis approach. These studies depended in large part on the recently determined high‐resolution structure of the G5BP dimer and cross‐correlations with physical‐chemical data available from other techniques. The approach was to systematically scan the full set of helical complexation parameters involved, based upon observed structural and orientational constraints, to determine those compatible with both the structure of the G5BP dimer and the orientational constraints, to determine those compatible with both the structure of the G5BP dimer and the overall dimensions of the full complex. This process was monitered throughout by close scrutiny of dimer‐dimer contacts and the use of hard‐copy and interactive graphics devices. Instead of the wide variety of possibilities that had been expected from such an approach, only one satisfactory assembly of DNA and G5BP dimers could be found. The results indicate that phage DNA will be wound to the outside of the helical protein ribbon that forms the core of intracellular complex at a density of five nucleotides per G5BP monomer. Bound DNA strands are positioned in two contiguous binding channels, which form as a consequence of the interactions of complexed G5BP dimers. These channels run just inside the outer extended β loops, composed of residue 20–30, and are separated by approximately 3.2 nm. The DNA phosphate backbone is bound at a substantially smaller radial distance (∼ 3.5 nm) than the maximum radius of the intracellular complex as a whole (∼ 4.5 nm) since bound DNA is embedded within these well‐defined binding channels. Our studies also indicate that a number of sterically unacceptable contacts, involving residues 38–42, prevent complexation of otherwise complementary dimer surfaces in the absence of nucleic acids. In the process of binding DNA, these residues change conformation thereby allowing self‐assembly of dimer units into a helical structure. We propose that these residues act as a two‐position sterochemical switch that allows or disallows complex formation in response to the absence or presence of DNA.
The mechanism of the C‐133 esterification step in the biosynthesis of bacteriochlorophyll a
Authors: AJAZ Abid A. , CORINA David L. , AKHTAR Muhammand
Published: March 2005

5‐Aminolaevulinate labelled with 18O at its C‐1 carboxy oxygen atoms was prepared and incorporated into bacteriochlorophyll aphytyl of Rhodopseudomonas sphaeroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum. The biosynthetic samples of the bacteriochlorophylls were separately processed to obtain their isoprenyl alcohol components from the C‐173 ester linkages and methanol from the C‐133 methoxycarbonyl group. Methods were developed for the quantification of the isotopic composition of the various alcohols (methanol, phytol, geranylgeraniol). It was shown that the hydroxyl oxygen atoms of all the three alcohols originated from one of the C‐1 oxygen atoms of the precursor 5‐aminolaevulinate. In the light of these results the in vivo mechanism for the O‐methylation reaction at C‐133 during the biosynthesis of the two species of bacteriochlorophylls is discussed.
Characterization of the binding sites for nimodipine and (−)‐desmethoxyverapamil in bovine cardiac sarcolemma
Authors: RUTH Peter , FLOCKERZI Veit , VON NETTELBLADT Egbert , OEKEN Jochem , HOFMANN Franz
Published: March 2005

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (−)‐desmethoxyverapamil were studied. The density of the nimodipine and (−)‐desmethoxyverapamil binding sites increased 8.3‐fold and 3.4‐fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin.

Nimodipine bound in the presence of 1 mM free calcium to a high‐affinity and a low‐affinity site with apparent Kd values of 0.35 ± 0.09 nM (n= 9) and 33 ± 6.0 nM (n= 9) and with apparent densities of 0.3 ± 0.05 pmol/mg (n= 9) and 8.2 ± 1.0 pmol/mg (n= 9). The binding to the high‐affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (−)‐isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism.

(−)‐Desmethoxyverapamil bound in the presence of 5mM EGTA to a high‐affinity and a low‐affinity site with apparent Kd values of 1.4 ± 0.3 nM (n= 6) and 171 ± 26 nM (n= 6) and with apparent densities of 0.16 ± 0.02 pmol/mg (n= 6) and 13.6 ± 2.7 pmol/mg (n= 6). The binding to both sites was inhibited by calcium with a half‐maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (−)‐isomers than for the (+)‐isomers. Nimodipine inhibited the binding of (−)‐desmethoxyverapamil by an apparent allosteric mechanism.

d‐cis‐Diltiazem inhibited non‐competitively the binding of (−)‐[3H]desmethoxyverapamil with a Ki of 3.7 μM. Diltiazem up to concentrations of 10 μM did not affect the amount of nimodipine bound at equilibrium at 20±C. However, but in agreement with this result, diltiazem decreased threefold at 20±C the dissociation and association rates for the high‐affinity nimodipine receptor. These rates were only marginally affected at 4±C and 37±C. d‐cis‐Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (−)‐desmethoxyverapamil with a Ka value of 1.6 μM.

The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low‐affinity site, but did not affect binding to the high‐affinity site. Binding of (−)‐desmethoxyverapamil was not affected by these adenosine uptake inhibitors.

These results show that bovine cardiac sarcolemma contains different high‐affinity receptors for dihydropyridines and phenylalkylamines which interact by allosteric mechanisms with each other and with diltiazem. The low‐affinity site for nimodipine may be identical with part of the adenosine transporter.
DNA methylation of bacterial viruses T3 and T7 by different DNA methylases in Escherichia coli K12 cells
Authors: KRÜGER Detlev H. , SCHROEDER Cornelia , REUTER Monika , BOGDARINA Irina G. , BURYANOV Yaroslav I. , BICKLE Thomas A.
Published: March 2005

We have investigated the susceptibility of the genomes of the related bacteriophages T3 and T7 to the three major DNA methyltransferases (EcoK, dam, dcm) of their host, Escherichia coli K12.

In vivo the EcoK host specificity enzyme only methylates the DNA of ocr– phages. This is due to an inhibition of the enzyme by the phage ocr+ gene product, which had previously been shown to be an inhibitor of the restriction endonuclease. EcoK‐specific DNA methylation protects the ocr– viruses after one growth cycle on these host cells against the action of corresponding restriction endonuclease EcoK.

Owing to the unique S‐adenosyl‐l‐methionine hydrolase (sam+) activity of the T3‐coded ocr+ protein, the T3 DNA is absolutely devoid of the methylated bases 6‐methylaminopurine and 5‐methylcytosine.

In contrast to this, T7 derivatives and sam– derivatives of T3 carry a small number of about 2–4 molecules 6‐methylaminopurine and 5‐methylcytosine per genome. The presence of 6‐methylaminopurine is due to dam methylation, though the majority of dam sites remain unmethylated. In vivo as well as in vitro the ocr+ protein has no influence on the activities of the dam and dcm methylase. The experiments gave some evidence for the existence of a second cytosine methylase in E. coli K12.

Besides dam and dcm recognition sites being undermethylated, their absolute number in T3 and T7 DNAs is far below the expected value. Moreover, one of the two dcm sites present in T7 (Studier strain) is missing in our T7 strain owing to a 1300‐base‐pair deletion in gene 0.7.
The amino acid sequence of a λ light chain presenting abnormal physicochemical and antigenic features
Authors: MIHAESCO Edith , ROY Jean‐Pierre , CONGY Nicole , PERAN‐RIVAT Liliane , MIHAESCO Constantin
Published: March 2005

The amino acid sequence of the light chain of a human monoclonal IgA1 (Mem) was established, in part by analogy with already known sequences. By homology its variable part was shown to belong to the VΔ1 subgroup while the isotype‐associated amino acid residues characterized it as Mcg+, Kern+ and Oz−. The normal primary structure of this chain was in contrast to its abnormal physical and antigenic properties: (a) its apparent molecular mass estimated by SDS/polyacrylamide gel electrophoresis, by gel filtration chromatography and by gradient ultracentrifugation was found to be lower by ∼ 10% than the values (23.5 kDa) of ‘normal’ light chain used as controls; (b) the Δ1 chain Mem, when tested in native state was not antigenically reactive. These abnormalities were reverted when the chain was treated with 8 M urea. These data suggest that the abnormal behaviour of Δ1 chain Mem is at a conformational level.
Cell‐free synthesis and co‐translational processing of the human asialoglycoprotein receptor
Authors: BREITFELD Philip P. , SCHWARTZ Alan L.
Published: March 2005

The human asialoglycoprotein receptor is a 46‐kDa membrane glycoprotein. It is initially synthesized as a 40‐kDa precursor species possessing two N‐linked high‐mannose oligosaccharides which is subsequently converted to the 46‐kDa mature product upon modification of its oligosaccharides of the complex form [Schwartz, A. L. & Rup, D. (1983) J. Biol. Chem. 258, 11249–11255]. To investigate further the biosynthesis of the human asialoglycoprotein receptor, we have utilized a cell‐free wheat germ translation system supplemented with dog pancreatic microsomal membranes and programmed with HepG2 and human liver RNA. The primary translation product of the human receptor is a single 34‐kDa species and this species is expressed throughout human fetal and adult development. The primary translation product possesses no cleavable signal peptide and is co‐translationally glycosylated to form the 40‐kDa precursor species. In addition, the human asialoglycoprotein receptor is co‐translationally inserted into microsomal membranes such that a 4‐kDa cytoplasmic tail is susceptible to trypsin digestion.
Three‐dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry
Authors: HOLAK Tadeusz A. , NILGES Michael , PRESTEGARD James H. , GRONENBORN Angela M. , CLORE G. Marius
Published: March 2005

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton‐distance restraints and 32 hydrogen‐bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root‐mean‐square difference of 0.20 ± 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3–12), 2 (residues 37–47) and 4 (residues 65–75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti‐parallel at an angle of ≊ 150° to helix 1. The smaller helix 3 (residues 56–63) is at an angle of ≊ 100° to helix 4.
Effects of metabolite binding to ribulosebisphosphate carboxylase on the activity of the Calvin photosynthesis cycle
Authors: PETTERSSON Gösta , RYDE‐PETTERSSON Ulf
Published: March 2005

The regulatory implications of the interaction of ribulosebisphosphate carboxylase with metabolites participating in the Calvin photosynthesis cycle has been examined by control analysis based on our recently described kinetic model for photosynthetic carbohydrate formation in the chloroplast of C3 plants. The results provide clear evidence that the Calvin cycle activity under conditions of light and CO2 saturation is insignificantly affected by the inhibition of ribulosebisphosphate carboxylase caused by metabolites such as 3‐phosphoglycerate, fructose 1,6‐bisphosphate, sedoheptulose 1,7‐bisphosphate, NADPH, and inorganic orthophosphate.

Due to the exceptionally high stromal concentration of the carboxylase, metabolite binding to the enzyme affects the Calvin cycle activity indirectly by reducing the pool of free orthophosphate and phosphorylated metabolites available for the cyclic reactions. This pool reduction corresponds typically to about 5 mM total phosphate and derives mainly from the binding of ribulose bisphosphate and orthophosphate.

Substantial amounts of the metabolites interacting with ribulosebisphosphate carboxylase are present in an enzyme‐bound form. The bound form of the Calvin cycle intermediates sedoheptulose bisphosphate, fructose bisphosphate, and ribulose bisphosphate typically accounts for about 70, 80, and 90%, respectively, of the total stromal concentration of the intermediate.
Transketolase from human leukocytes
Authors: MOCALI Alessandra , PAOLETTI Francsco
Published: March 2005

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM‐cellulose by specific elution with a D‐xylulose‐5‐phosphate/D‐ribose‐5‐phosphate mixture and the isolated product exhibted a specific activity of about 10 units/mg protein at 37°C. Transketolase preparations are contamination‐free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D‐xylulose 5‐phosphate and D‐ribose 5‐phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa.

The isoelectrophoretic heterogeneity of transketollase was assesed either by activity staining or immunovisualization with anit‐transketolase antisera, previously induced in rabbits. These techniqus yielded two practically overlapping patterns consisting of 6–8 distinct bands within a pI range of 6.5–8.5. Both pure and crude transketolase preparations showed a similar heterogenous profil, thus confirming the stability of the enzyme throughout purification.

The occurrenc of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.
Monoclonal antibodies against antithrombin III
Authors: KNOLLER Sarah , SAVION Naphtali
Published: March 2005

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin‐binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1–393, at a site different from the active site since it recognizes antithrombin III and antithrombin‐III–thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin‐enhanced antithrombin III activity when added to the antithrombin‐III–heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282–393, close to the active site. It does not recognize antithrombin‐III–thrombin complexes by solid‐phase redioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect. but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1–393, and its recognition of antithrombin III or antithrombin‐III–thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activites. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339–393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure activity relationships in the antithrombin III molecule.
Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF‐hand
Authors: JOHANSSON Charlotta , BRODIN Peter , GRUNDSTRÖM Thomas , THULIN Eva , FORSÉN Sture , DRAKENBERG Torbjörn
Published: March 2005

The genes for four mutant proteins from calbindin D9k, all with mutations in the N‐terminal Ca2+‐binding domain (pseudo EF‐hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF‐hand is an attempt to turn this site into a structure resembling an archetypal EF‐hand, with its characteristic 113Cd‐NMR shift (–80 to −110 ppm) and high calcium‐binding constants, whereas the C‐terminal Ca2+‐binding site (EF‐hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF‐hands with a lower calcium‐binding constant and a higher calcium off‐rate to the pseudo EF‐hand than the wild‐type protein. From the results obtained it is obvious that proline 20 in the pseudo EF‐hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF‐hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium‐binding properties of the unmodified site virtually unchanged.
Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion
Authors: BRÜNE Bernhard , SCHMIDT Klaus‐Ulrich , ULLRICH Volker
Published: March 2005

Human platelet soluble guanylate cyclase activity was studied with respect to the function of its heme‐containing regulatory subunit. As an enzyme source, the 10000xg supernatant was used and, since its specific activity proved to be too low for inhibition studies, also a partially purified preparation was employed. The partially purified enzyme was stimulated about 2.5‐fold by carbon monoxide and this effect was abolished by illumination with visible light. Sodium nitroprusside also increased the basal activity about fourfold, which, however, is much less than the > 100‐fold stimulation seen with the supernatant. Superoxide anions generated by the xanthine/xanthine‐oxidase system were strongly inhibitory in the enriched preparation as well as in the CO‐stimulated platelet supernatant (median effector concentration = 0.1 mU/ml). Unlike CO and NO, the effect of superoxide cannot be mediated through the heme‐containing regulatory subunit, since heme‐free enzyme, which could not be activated by NO or CO, was inhibited to the same extent as the heme‐containing enzyme. Superoxide dismutase did not influence the basal activity, but resulted in a synergistic stimulation in the presence of CO. When Mn2+ as a cofactor, the basal activity was higher but superoxide could not inhibit the enzyme, possibly due to the superoxide‐dismutase‐like activity of Mn2+. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with endothelium‐derived relaxing factor, recently identified as NO, could form a physiologically relevant regulatory effector system.
Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis
Authors: KHMELNITSKY Yuri L. , MOZHAEV Vadim V. , BELOVA Alla B. , SERGEEVA Maria V. , MARTINEK Karel
Published: March 2005

The process of reversible denaturation of several proteins (α‐chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N‐methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so‐called ‘bad’ solvents; reasons for the occurrence of ‘bad’ solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well‐known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water‐immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.
Late Paleozoic adakites and Nb‐enriched basalts from northern Xinjiang, northwest China: Evidence for the southward subduction of the Paleo‐Asian Oceanic Plate
Authors: Zhang Haixiang , Niu Hecai , Sato Hiroaki , Yu Xueyuan , Shan Qiang , Zhang Boyou , Ito Jun’ichi , Nagao Takashi
Published: March 2005

Abstract  Volcanic rocks consisting of adakite and Nb‐enriched basalt are found in the early Devonian Tuoranggekuduke Group in the northern margin of the Kazakhstan‐Junggar Plate, northern Xinjiang, northwest China. The geochemical characteristics of the andesitic and dacitic rocks in this area resemble that of adakites. The relatively high Al2O3, Na2O and MgO content and Mg♯ values indicate that the adakites were generated in relation to oceanic slab subduction rather than the partial melting of basaltic crust. A slightly higher SrI and a lower ɛNd(t = 375 Ma) compared to adakites of mid‐oceanic ridge basalt (MORB) imply that slab sediments were incorporated into these adakites during slab melting. The Nb‐enriched basalt lavas, which are intercalated in adakite lava suite, are silica saturated and are distinguished from the typical arc basalts by their higher Nb and Ti content (high field strength element enrichment). They are derived from the partial melting of the slab melt‐metasomatized mantle wedge peridotite. Apparently, positive Sr anomalies and a slightly higher heavy rare earth element content in these adakites compared to their Cenozoic counterparts indicate that the geothermal gradient in the Paleo‐Asian Oceanic subduction zone and the depth of the Paleo‐Asian Oceanic slab melting are between those of their Archean and Cenozoic counterparts. The distribution of the adakites and Nb‐enriched basalts in the northern margin of the Kazakhstan‐Junggar Plate, northern Xinjiang, indicates that the Paleo‐Asian Oceanic Plate subducted southward beneath the Kazakhstan‐Junggar Plate in the early Devonian period.
Top Incomes in the UK Over the 20th Century
Authors: Atkinson A. B.
Published: March 2005

Recent changes in the distribution of income need to be placed in historical context. The paper provides new evidence about the evolution of top incomes in the UK over the 20th century. Making use of published tabulations of the income tax statistics, and of microdata for recent years, we construct estimates of the shares of top income groups, giving for the first time an annual time series for gross incomes that spans more than 90 years. The paper pays particular attention to the problems of data construction and of the interpretation of tax-based evidence. The resulting statistics have evident limitations but throw light on periods, such as that between the First and Second World Wars, for which there is little other empirical material. The results bring out clearly how the major equalization of the first three-quarters of the century in the UK has been reversed, taking the shares of the top income groups back to levels of inequality found 50 years ago. A similar U-shaped pattern is found for the USA, but the post-war experience of France is different from that in the UK.
Estimating Parliamentary Composition Through Electoral Polls
Authors: Udina Frederic , Delicado Pedro
Published: March 2005

All electoral systems have an electoral formula that converts proportions of votes into Parliamentary seats. Pre-electoral polls usually focus on estimating proportions of votes and then apply the electoral formula to give a forecast of Parliamentary composition. We describe the problems that arise from this approach: there will typically be a bias in the forecast. We study the origin of the bias and some methods for evaluating and reducing it. We propose a bootstrap algorithm for computing confidence intervals for the allocation of seats. We show, by Monte Carlo simulation, the performance of the proposed methods using data from Spanish elections in previous years. We also propose graphical methods for visualizing how electoral formulae and Parliamentary forecasts work (or fail).
Breaking the Boundaries: Geographical Approaches to Integrating 200 Years of the Census
Authors: Gregory Ian N. , Ell Paul S.
Published: March 2005

The census and similar sources of data have been published for two centuries so the information that they contain should provide an unparalleled insight into the changing population of Britain over this time period. To date, however, the seemingly trivial problem of changes in boundaries has seriously hampered the use of these sources as they make it impossible to create long run time series of spatially detailed data. The paper reviews methodologies that attempt to resolve this problem by using geographical information systems and areal inter-polation to allow the reallocation of data from one set of administrative units onto another. This makes it possible to examine change over time for a standard geography and thus it becomes possible to unlock the spatial detail and the temporal depth that are held in the census and in related sources.
Age at First Marriage in Malawi: A Bayesian Multilevel Analysis Using a Discrete Time-to-Event Model
Authors: Manda Samuel , Meyer Renate
Published: March 2005

The paper presents a hierarchical discrete time survival model for the analysis of the 2000 Malawi Demographic and Health Survey data to assess the determinants of transition to marriage among women in Malawi. The model explicitly accounts for the unobserved heterogeneity by using family and community random effects with cross-level correlation structure. A nonparametric technique is used to model the base-line discrete hazard dynamically. Parameters of the model are computed by using a Markov chain Monte Carlo algorithm. The results show that rising age at marriage is a combination of birth cohort and education effects, depends considerably on the family and to some extent on the community in which a woman resides and the correlation between family and community random effects is negative. These results confirm a downward trend in teenage marriage and that raising women's education levels in sub-Saharan Africa has the beneficial effect of increasing age at marriage, and by implication reducing total fertility rates. The negative correlation between family and community random effects has policy implications in that targeting communities with an intervention to increase age at first marriage may not necessarily yield reduced fertility levels in individual families. A campaign that is geared towards individual families would achieve the desired goals. Overall, the findings point to the need for the Government in Malawi to enact public policies which are geared at vastly improving women's education at higher levels. The variation in marriage rates over families poses problems in delivering the policy, since particular policies must be devised for specific groups of families to accomplish the required social and health objectives.
Trimethylaminuria (fish‐odour syndrome) and oral malodour
Authors: Mitchell SC
Published: March 2005

A small but important percentage of oral malodour cases have an extra‐oral aetiology and certain of these fall into the category of ‘blood‐borne halitosis’. Odoriferous substances generated within the body and transported to the lungs via the circulatory system may, if sufficiently volatile, leave with the exhaled air and impart a foetid odour to the breath. The aliphatic tertiary amine, trimethylamine, is such a volatile compound that is generated to excess in patients with a metabolic disorder known as trimethylaminuria (fish‐odour syndrome). This article highlights this condition and draws attention to its potential role in the causation of recalcitrant oral malodour.
Comparison of ninhydrin method of detecting amine compounds with other methods of halitosis detection
Authors: Iwanicka‐Grzegorek K , Lipkowska E , Kepa J , Michalik J , Wierzbicka M.
Published: March 2005

Objectives:  Halitosis is a result of overactivity of Gram‐negative bacteria for which protein amino acids are the major source of energy. Therefore, statistical correlation between concentrations of volatile sulphur compounds (VSCs) and low‐molecular‐weight amines should be expected.

Materials and methods:  Eighty‐four patients suffering from halitosis and 40 healthy volunteers aged 20–62 years (average 39.7) participated in the study. In all subjects low‐molecular‐weight amines were evaluated by the ninhydrin method. Patients with halitosis were randomized into treatment groups. Zinc tablets, tablets and mouthwash containing chlorhexidine or lyophilized lactic acid‐forming bacteria were used.

Results:  Analysis showed that the level of amines was highest in subjects with halitosis (0.39, s.d. ± 0.06, P < 0.001) and correlated significantly with results of VSC measurement and organoleptic scores (P < 0.001). Reduction of amine levels after treatment was statistically significant (P < 0.001). Three months following treatment the amine levels began to increase (0.37, s.d. ± 0.05). The amine levels in healthy controls were lower (0.29, s.d. ± 0.07) and remained at a stable level.

Conclusion:  The salivary amine levels significantly correlated with VSC levels and organoleptic scores. Therefore, the ninhydrin method of detecting salivary amines may be an alternative or additional method of diagnosing halitosis. This method may also be used to evaluate treatment efficacy.
Clinical effectiveness of a triclosan/copolymer/sodium fluoride dentifrice in controlling oral malodor: a 3‐week clinical trial
Authors: Hu D , Zhang YP , Petrone M , Volpe AR , DeVizio W , Giniger M
Published: March 2005

Objective:  The purpose of the study was to compare the effectiveness of a dentifrice containing 0.3% triclosan, 2% polyvinylmethylether/maleic acid (PVM/MA) copolymer, 0.243% sodium fluoride (TCF) to a commercially‐available dentifrice containing 0.243% sodium fluoride (control) for the management of oral malodor in a 3‐week, randomized double‐blind, longitudinal clinical trial.

Methods:  A panel of four expert judges used a nine‐point hedonic scale to evaluate breath odor using a protocol designed in accordance with the ADA Draft Acceptance Program Guideline for Products Used in the Management of Oral Malodor. Following a baseline evaluation, prospective subjects with hedonic scores above the threshold value for unpleasant breath were stratified by score and randomized into two treatment groups. Subjects brushed their teeth for 1 min with their assigned dentifrice after which they were scored for oral malodor at 1.5, 4 and 12 h. They then used their assigned dentifrice, twice a day, for 3 weeks. Before oral malodor evaluations, the subjects refrained from eating odorigenic foods, using mouthrinses and breath mints and from performing dental hygiene procedures.

Results:  Eighty‐one adult male and female subjects completed the study. The baseline hedonic scores for the TCF and control dentifrices were 7.80 and 7.84, respectively, corresponding to unpleasant breath. The final mean oral malodor scores for the TCF dentifrice differed significantly from the baseline and control values (P < 0.05) for every time point examined (1.5‐, 4‐, 12‐h and 1‐, 2‐ and 3‐week intervals). The mean final breath scores for the TCF dentifrice group were 3.06, 3.48, 3.42, 3.66, 3.41 and 3.36, respectively, for each time point. These scores correspond to pleasant breath. Conversely, the control dentifrice group scored at levels either above 5.0 (before 12 h) or above 7.0 (after 12 h) which corresponded to neutral or unpleasant breath.

Conclusion:  In conclusion, the results of this double‐blind clinical study clearly indicate that a dentifrice containing triclosan/copolymer/NaF provides effective control of oral malodor for up to 12 h.
Effect of a triclosan/PVM/MA copolymer/fluoride dentifrice on volatile sulfur compounds in vitro
Authors: Pilch S , Williams MI , Cummins D
Published: March 2005

Objective:  The objective of the investigation was to document the in vitro efficacy of a triclosan/PVM/MA copolymer/fluoride (TCF) dentifrice against the formation of volatile sulfur compounds (VSC) as well as the growth of H2S‐producing bacteria. Clinical studies using organoleptic judges, gas chromatography, or a portable sulfide monitor have generally been employed in the assessment of treatments for the control of oral malodor. However, these studies are not appropriate for screening purposes because of the expense and time required.

Methods:  An in vitro method was developed for the purpose of screening new compounds, agents or formulations for their ability to control VSC formation and for determining bio‐equivalence of efficacy when implementing changes in existing formulations. The method combines basic microbiological methods, dynamic flow cell techniques and head space analysis. The in vitro VSC method was validated by comparing the efficacy of two dentifrices containing TCF with a control fluoride dentifrice as the TCF products have been clinically proven to control oral malodor.

Results:  In the validation studies, the TCF‐containing dentifrices were significantly better (P < 0.05) than the control dentifrice in inhibiting VSC formation and reducing H2S‐producing bacteria. For example, when compared with baseline, the TCF dentifrices reduced VSC formation between 42 and 49% compared with the control dentifrice which reduced VSC formation 3%. There was no significant difference (P > 0.05) between the two TCF dentifrice formulations.

Conclusion:  Using an in vitro breath VSC model, it has been demonstrated that two variants of a dentifrice containing triclosan, PVM/MA copolymer and fluoride have efficacy that is significantly better than a control fluoridated dentifrice and that there is no significant difference between the triclosan/PVM/MA copolymer/fluoride dentifrice variants.
Relationship between the SF‐36 questionnaire and patient's satisfaction following halitosis therapy
Authors: Kishi M , Abe A , Yonemitsu M
Published: March 2005

Objective:  To assess the health‐related quality of life (HRQOL) of patients complaining of halitosis at their first visit and at a later time when their complaint had diminished following therapy, using a self‐administered questionnaire, the Medical Outcome Study Short Form‐36 (SF‐36). The aim of this study was to examine the relationship between HRQOL of patients before and after self‐reported disappearance of their complaint following oral hygiene improvements for halitosis.

Subjects and methods:  Seventy patients of our special clinic for halitosis served as subjects. At the first visit, each completed the SF‐36 before determination of volatile sulfur compound (VSC) concentration in mouth air. After excluding dropouts, the same measurements were performed for subjects whose self‐reported complaint had disappeared following oral hygiene therapy.

Results and discussion:  At the initial visit, SF‐36 scale scores for general health, vitality, social functioning, role‐emotion, and mental health were significantly lower when compared with the national averages in Japan. For subjects with self‐reported disappearance of complaint, only social functioning rose significantly among SF‐36 scores at the end of the study. These results suggest that an awareness of improvement in social life could be related to patient's satisfaction with halitosis oral hygiene therapy.