bluuebunny/crossref_metadata_2025_split huggingface dataset:
Overproduction of staphylokinase in Escherichia coli and its characterization
Authors: SAKO Tomoyuki 
Published: March 2005

A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylo‐coccus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage λ PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60‐fold and the 15.5‐kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5‐kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis.By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM‐cellulose column chromatography. Two species of staphylokinase were identified after CM‐cellulose column chromatography. Although their isoelectric points and NH2‐terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2‐terminal portion of staphylokinase is not important for its activity.
The complete 30‐base‐pair origin region of bacteriophage φ X174 in a plasmid is both required and sufficient for in vivo rolling‐circle DNA replication and packaging
Authors: FLUIT Adriaan C. , BAAS Pieter D. , JANSZ Hendrik S. 
Published: March 2005

The origin of replication of the isometric single‐stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling‐circle DNA replication and packaging of single‐stranded (ss) plasmid DNA into phage coats in φ X 174 or G4‐phage‐infected cells.This system was used to study the nucleotide sequence requirements for rolling‐circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30‐base‐pair (bp) origin region of φ X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30‐bp origin region and plasmids carrying the 30‐bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of φ X 174 replicative‐form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30‐bp origin region and that no contribution is made by sequences which immediately surround the origin region in the φ X 174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30‐bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the φ X 174 and G4 systems respectively).Previous studies [Fluit, A. C., Baas, P. D., van Boom, J. H., Veeneman, G. H. and Jansz, H. S. (1984) Nucleic Acids Res. 12, 6443–6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by φ X 174 gene A protein. Moreover, Brown et al. [Brown, D. R., Schmidt‐Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402–8412] have shown that omission of the last 2 bp of the origin region does not interfere with φ X 174 rolling‐circle DNA replication in vitro.Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for φ X 174 phage DNA replication and morphogenesis.
Reconstitution and fusogenic properties of Sendai virus envelopes
Authors: HARMSEN Marco C. , WILSCHUT Jan , SCHERPHOF Gerrit , HULSTAERT Caesar , HOEKSTRA Dick 
Published: March 2005

Sendai virus membranes were reconstituted by detergent dialysis, using the non‐ionic detergents Triton X‐100 and octyl glucoside. Membrane reassembly was determined by measuring the surface‐density‐dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co‐reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X‐100 with the reconstituted membrane was revealed by an artificial detergent‐effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer.Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.
Natural‐abundance 13C nuclear magnetic resonance studies of regulation and overproduction of l‐lysine by Brevibacterium flavum
Authors: INBAR Livia , KAHANA Zvi E. , LAPIDOT Aviva 
Published: March 2005

Natural‐abundance 13C NMR spectroscopy has been used to study the metabolism of the l‐lysine‐producing bacterium, Brevibacterium flavum. Relationships of biomass formation, precursor uptake, and product excretion, as a function of culture medium, oxygen supply and specific cell membrane permeability, were rapidly determined using 67.89‐MHz 13C NMR. The induction of lysine production throughout the growth cycle was studied. Intracellular and extracellular levels of free metabolites and unconsumed precursor were quantitatively measured as a function of growth culture conditions. Limited availability of oxygen resulted in accumulation and excretion of unfavorable products: lactate, succinate, alanine and valine. However, under optimal aeration conditions l‐lysine was the sole metabolite detected extracellularly. Various important long‐lived intermediates and storage compounds were detected in the intact cells (by NMR measurements). Carbon resonances of carbohydrates and amino acids were resolved and easily identified. Of particular interest are those of trehalose carbons, a storage carbohydrate.Natural‐abundance 13C NMR spectroscopy seems most suitable for biotechnological processes where high concentrations of intermediates and end‐products can be observed. We anticipate that this approach will be employed to screen overproducing bacterial strains.
Lateral proton conduction at a lipid/water interface
Authors: PRATS Michel , TOCANNE Jean‐François , TEISSIÉ Justin 
Published: March 2005

Fast lateral proton conduction along the lipid/water interface has recently been experimentally demonstrated in our laboratory [Teissié, J., Prats, M., Soucaille, P. & Tocanne, J. F. (1985) Proc. Natl Acad. Sci. USA, in the press].The present study gives a more precise description of the way various physical parameters can affect this process. The dependence of the distance covered by the proton on time is demonstrated to be quadratic. Increasing the speed of stirring in the injection compartment or the amount of injected acid or the contact between the monolayer and the acidic subphase increased the efficiency of the proton transfer. Raising the strength of the buffer in the bulk phase inhibited proton conduction.Results from experiments where the transfer of protons from the bulk phase to the interface was modified, suggested the occurrence of an ‘energy barrier’ limiting the access of protons from the bulk phase to the lipid polar head region.
Hysteretic behaviour of citrate synthase
Authors: LÖHLEIN‐WERHAHN Gerlinde , GOEPFERT Petra , EGGERER Hermann 
Published: March 2005

Citrate specimens derived from chiral acetates were converted to the CoA derivatives. These were reconverted with citrate synthase to citrates under conditions of either predominating hydrolytic burst or predominating steady‐state period. The stereochemical purity of substrates and products was determined.
Reversal of the synthase condensation step occurs under both conditions but is markedly increased during the steady‐state period. The results indicate that citryl‐CoA‐derived acetyl‐CoA and oxaloacetate create the steady‐state conditions.
The hydrolase state and the ligase/lyase state of the synthase predominate under burst and steady‐state conditions, respectively. This result indicates a conversion of the hydrolase state into the ligase/lyase state during the transition.
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.
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 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.
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.
Top Incomes in the UK Over the 20th Century
Authors: Atkinson A. B. 
Published: March 2005

SummaryRecent 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

SummaryAll 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

SummaryThe 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.