bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic and Steady‐State Investigations of Solute Accumulation in Bacterial Membranes by Continuously Monitoring the Radioactivity in the Effluent of Flow‐Dialysis Experiments
Authors: HELLINGWERF Klaas J. , KONINGS Wil N. 
Published: March 2005

The flow‐dialysis technique for studies of solute accumulation by membrane preparations has been made more suitable for routine measurements by recording continuously the radioactivity in the effluent of a flow‐dialysis vessel with a homogeneous flow‐monitoring device for β‐emitters. This modification not only decreases the time and cost of a flow‐dialysis experiment but also allows the investigator to react directly on the outcome of his experiments.Analysis of the kinetics of this automated flow‐dialysis system shows that this technique can also be used for the determination of the rate of uptake of solutes into bacterial membranes. This has been confirmed in Rhodopseudotomonas sphaeroides by comparing the results of Rb+ and inorganic phosphate uptake studies performed by automated flow‐dialysis and by the conventional filtration procedure. This application has the limitation that solute uptake has to proceed linearly for a period of about five times the half‐time of the response of the flow‐dialysis system.The two described applications make automated flow‐dialysis very well‐suited for experiments on the bioenergetics and regulation of solute uptake into bacterial membranes. Both driving force and rate of solute uptake can now be determined in one experiment.
The Reversible Dehydration of (R)‐2‐Hydroxyglutarate to (E)‐Glutaconate
Authors: BUCKEL Wolfgang 
Published: March 2005

During fermentation with whole cells of Acidaminococcus fermentans or Clostridium microsporum the pro‐3S hydrogen of (R)‐2‐hydroxyglutarate or of its precursor (S)‐glutamate is eliminated stereospecifically. Since (E)‐glutaconate but not its Z isomer is fermented by whole cells or cell‐free extracts of A. fermentans, the overall dehydration of (R)‐2‐hydroxyglutarate to (E)‐glutaconate can be described as syn.
The fermentation of (E)‐glutaconate required acetyl phosphate, CoA and NAD, that of (S)‐glutamate or (R)‐2‐hydroxyglutarate additionally MgCl2, FeSO4 and dithioerythritol. The fermentations of all three substrates were inhibited by avidin and stimulated by biotin.
The hydration of (E)‐glutaconate was measured enzymically by the formation of (R)‐2‐hydroxyglutarate. The dehydration of the hydroxy acid was assayed by the release of 3HOH from (2R)‐2‐hydroxy[3‐3H]glutarate. Optimum conditions were found by activation of the cell‐free extract with MgCl2, FeSO4, dithioerythritol, acetyl phosphate and NADH followed by the reaction which only required acetyl phosphate and CoA as cofactors. Activation and reaction had to be performed anaerobically.
The dehydration was inhibited by 2mM azide, 1mM arsenate, 1mM hydroxylamine, 20μM dinitrophenol or 10μM carbonylcyanide p‐trifluoromethoxyphenylhydrazone.
It is concluded that the actual substrates of the dehydration are the corresponding thiol esters. The data indicate a catalytical phosphorylation during the reaction.
An Electron Microscopic Analysis of rRNAs and tRNAs and of the Assembly in vitro of the 50‐S Subunits from Escherichia coli Ribosomes
Authors: SIEBER Gerhard , TESCHE Bernd , NIERHAUS Knud H. 
Published: March 2005

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow‐cast technique. The 16‐S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23‐S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ± 4.5 nm. Different 23‐S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5‐S RNA shows an extended rod‐like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N‐acetylphenylalanyl‐tRNAPhe respectively.
Analysis of the assembly in vitro of the ribosomal 50‐S subunit. The assembly of the 50‐S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two‐step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585–599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly‐(Phe)‐synthesizing activity in the presence of 30‐S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50‐S subunit in vitro. The results indicate that the reconstitution intermediate stage RI*50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles.
Metabolism of 4‐O‐Methyl‐N‐acetylneuraminic Acid a Synthetic Sialic Acid
Authors: BEAU Jean‐Marie , SCHAUER Roland 
Published: March 2005

4‐O‐Methyl‐N‐acetylneuraminic acid shows a strong positive periodate‐thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published.
An efficient preparation of a tritium‐labelled 4‐O‐methyl‐N‐acetylneuraminic acid, with high specific radioactivity, by an oxymercuration‐demercuration procedure is presented.
Sialyltransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP‐N‐acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP‐4‐O‐methyl‐N‐acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates.
The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N‐acetylneuraminic acid or 4‐O‐methyl‐N‐acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl‐plague virus neuraminidase. In contrast, 4‐O‐methyl‐N‐acetylneuraminic acid‐fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase.
Free 4‐O‐methyl‐N‐acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate‐lyase. It does not inhibit the enzymic cleavage reaction of N‐acetylneuraminic acid.
The influence of a substitution at C‐4 of neuraminic acid on the enzymatic reaction mechanisms is discussed.
Purification and DNA‐Binding Properties of RNA Polymerase from Bacillus subtilis
Authors: GIACOMONI Paolo U. 
Published: March 2005

Four RNA‐polymerizing activities having different subunit composition can be purified from uninfected and from SPO1‐infected Bacillus subtilis.Lysozyme and sodium deoxycholate are used for lysing the cells. Polymin P is used for precipitating nucleic acids and DEAE‐cellulose chromatography allows separation of enzymatic activity from the residual Polymin P. After these common steps, one can purify core +σ+δ by chromatography on single‐stranded DNA‐agarose followed by gel filtration while pure core +σ can be obtained by chromatography on double‐stranded DNA cellulose. Core +δ is obtained by high‐salt sucrose/glycerol gradient centrifugation. The host enzyme modified by the product of gene 28 of phage SPO1 can be purified from SPO1 infected cells by chromatography on DNA cellulose (or DNA agarose) followed by chromatography on phosphocellulose.The pH and salt dependance of the initial rate of RNA synthesis of core +σ has been investigated using SPO1 and SPP1 DNA as templates. The optimum pH for the initial rate of transcription is 8.2 at 30°C in 50 mM N,N‐bis(2‐hydroxyethyl)glycine buffer, and the optimum Na+ concentration is between 0.1 and 0.15 M.The kinetics of formation and of dissociation of non‐filterable complexes between SPP1 DNA and core +σ have been analyzed at different cationic concentrations. The value of the rate constant of dissociation in 0.1 M NaCl at 30°C is kd= 2.16 × 10−4 s−1. The value of the rate constant of association, under the same conditions, is ka= 5.5 × 108 M−1 s−1; this value is compatible with a diffusion‐controlled reaction for promoter selection.
Extraction and Translation of Collagen mRNA from Fetal Calf Skin
Authors: KAUFMANN Rolf , BELAYEW Alexandra , NUSGENS Betty , LAPIÈRE Charles M. , GIELEN Jacques E. 
Published: March 2005

RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)‐rich RNA was separated by oligo(dT)‐cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell‐free system, unfractionated guanidine‐hydrochloride‐extracted poly(A)‐rich RNA directed the synthesis of two collagenase‐sensitive protein bands, while phenol‐extracted poly(A)‐rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/urea/polyacrylamide gel, these proteins were identified with procollagen α1(I) and procollagen α2. Inhibition of translation by phenol‐extracted poly(A)‐rich RNA with a sedimentation coefficient lower than 25 S was also observed.Guanidine‐hydrochloride‐extracted poly(A)‐rich RNA from fetal calf skin directed the synthesis of three distinct collagenase‐sensitive proteins in the micrococcal‐nuclease‐digested rabbit reticulocyte cell‐free system; these seemed to correspond to procollagen α1(I), procollagen α2 and procollagen α1(III).
Messenger RNA for Ribosomal Proteins in Xenopus laevis Oocytes
Authors: PIERANDREI‐AMALDI Paola , BECCARI Elena 
Published: March 2005

Xenopus laevis oocytes (stage II—III) incorporate [35S]methionine into ribosomal proteins identifiable by two‐dimensional electrophoresis. Poly(A)‐rich RNA prepared from this source has been translated in a wheat germ cell‐free system. Among the products synthesized in vitro 30 are clearly identifiable as ribosomal proteins. Some are not individually resolved, whereas a few of them may lack methionine or may not be correctly processed after the synthesis in vitro.An enrichment of our mRNA preparation for ribosomal protein activity can be obtained by subsequent passages on oligo(dT)‐cellulose columns and by selection of a 10–16‐S fraction by sucrose gradient sedimentation. We estimate that messenger RNA for ribosomal proteins represents 10–20% of this RNA preparation and can now be utilized for molecular cloning procedures.
Amplification of a Mitochondrial DNA Sequence in the Cytoplasmically Inherited ‘Ragged’ Mutant of Aspergillus amstelodami
Authors: LAZARUS Colin M. , EARL Alison J. , TURNER Geoffrey , KÜNTZEL Hans 
Published: March 2005

A comparison has been made between mtDNA of the cytoplasmically inherited ‘ragged’ mutant of Aspergillus amstelodami and that of the wild‐type strain. Ragged mitochondria contain both the wild‐type mitochondrial genome and several large DNA molecules which are not cleaved by the restriction endonucleases BumHI, HaeIII, HhuI, HindII, HindIII, PstI and MboI, but are converted by either EcoRI or HpaII into a single 820–840 base‐pair fragment. Restriction analysis and molecular hybridization data indicate that this fragment contains sequences of wild‐type mtDNA located within a 1200‐base‐pair segment of the 40500‐base‐pair genome, for which a basic restriction map has been deduced.It is concluded that in the ragged mutant a small segment of wild‐type mtDNA has been amplified as tandem repeats, which is reminiscent of the Rho− petite phenotype of yeast. The results are discussed in relation to the phenomenon of senescence in Podospora unserinu.
Analysis of the Population of Native 40‐S Ribosomal Subunits in Mouse Plasmacytoma Cells Grown in Suspension Culture
Authors: VANGDAL Eivind , EIKHOM Thor S. 
Published: March 2005

The native ribosomal subunits of the mouse plasmacytoma cell line MPC‐11 were fractionated by CsCl gradient centrifugation into seven classes of particles designated p1, p2, p3, p4, p5, p6 and p7, which possessed densities of approximately 1.38, 1.43, 1.46, 1.49, 1.50, 1.51 and 1.53 g/cm3 respectively. The RNA was analysed by oligo(dT)‐cellulose chromatography and gel electrophoresis.In both well‐fed and starved cells p3 was generally the dominating class of particles. During metabolic shift‐up conditions, however, there was a great increase of p6 particles. Transfer of material containing rRNA and mRNA into this region of the CsCl gradient indicates the accumulation of an initiation complex containing mRNA.During metabolic shift‐down conditions a decrease of p6 particles and an increase of p2 and p3 particles was observed. The p2 fraction was rich in mRNA and may contain considerable quantities of messenger ribonucleoprotein particles. A substantial fraction of newly synthesised mRNA‐containing material in starved cells banded in fact in the p2 region of the CsCl gradients. Circumstantial evidence indicates the existence of a transient 40‐S initiation complex which bands in the p2‐p3 region of the gradients.Double‐labelling experiments demonstrated that radioactivity in 40‐S subunits first appeared in precursor particles with a density of 1.49–1.50 g/cm3 and only later in p3 and p6 particles. p7 particles were detected in starved cells. They were slowly labelled and were presumably derived from old ribosomes following completion of the polysome cycle.
The Ganglioside Content of the Milk Fat‐Globule Membrane and the Mouse Mammary‐Tumour Virus Isolated from the Milk of Infected Mice
Authors: GOSSELIN‐REY Cecile , GOSSELIN Luc , CALBERG‐BACQ Claire‐Michelle , FRANÇOIS Camille , KOZMA Sara , OSTERRIETH Paul M. , VAN DESSEL Guido 
Published: March 2005

The milk fat‐globule membrane and the mouse mammary‐tumour virus isolated from the milk of infected Swiss mice have been investigated for their content in gangliosides. When compared on the lipid phosphorus basis, viral envelope is found to contain more than twice as much lipid‐bound sialic acid as fat‐globule membrane.The ganglioside patterns of these two structures appear rather similar, except for the occurrence in fat‐globule membrane of a low ganglioside homolog, presumably GM2, not detected in viral envelope.A common and dominant trait is the presence in both structures, as the main ganglioside, of a component which has been so far characterized as a disialoganglioside, having the same neutral glycolipid moiety as GD1a, but with both sialic acid residues displaying to Clostridium perfringens and Vibrio cholerae neuraminidase, the susceptibility typical of terminal sialic acid residues.
The Mechanism of Action of Eukaryotic Initiation Factor 4C in Protein Synthesis
Authors: THOMAS Adri , GOUMANS Hans , VOORMA Harry O. , BENNE Rob 
Published: March 2005

Binding of eukaryotic initiation factor 4C (eIF‐4C) to a 40‐S initiation complex could be detected by analysis of assay mixtures for 40‐S initiation complex formation on columns of Sepharose 6B and on sucrose gradients. On Sepharose 6B columns an almost stoichiometric binding of [14C]eIF‐4C was observed, provided that elF‐1, elF‐2, eIF‐3, Met‐tRNAf and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 μM edeine was an additional prerequisite for stable binding of eIF‐4C.The presence of eIF‐4C on the 40‐S initiation complex resulted in a pronounced increase of its stability, which might explain the observed stimulatory effect of the factor on the binding of Met‐tRNAf into initiation complexes.The interaction between eIF‐4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since eIF‐4C was capable of dissociating 80‐S ribosomes into subunits. Protein synthesis in a ‘pH‐5’ assay was almost totally dependent on the addition of eIF‐4C, when the assay was performed with purified, nearly homogeneous, initiation factors.
Purification of Haemagglutinin and Neuraminidase from Influenza Virus Strain 3QB and Isolation of a Peptide from an Antigenic Region of Haemagglutinin
Authors: AITKEN Alastair , HANNOUN Claude 
Published: March 2005

Methods were developed for the purification of the surface, membrane‐bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin.Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16‐residue peptide with amino‐terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.
Amino Acid Sequence of the Proteolipid Subunit of the Proton‐Translocating ATPase Complex from the Thermophilic Bacterium PS‐3
Authors: HOPPE Jürgen , SEBALD Walter 
Published: March 2005

The proteolipid subunit of the ATPase complex was identified in whole membranes of the thermophilic bacterium PS‐3 by means of a covalent modification with the 14C‐labelled inhibitor dicyclohexylcarbodiimide. The proteolipid could be purified from the membrane in free and carbodiimide‐modified form by extraction with chloroform/methanol and subsequent carboxymethyl‐cellulose chromatography in mixtures of chloroform/methanol/water.The complete amino acid sequence of the 72‐residue polypeptide could be determined by automated solid‐phase Edman degradation of the whole protein, and of fragments obtained after cleavage with cyanogen bromide and N‐bromosuccinimide. Chemical cleavages and separations of the resulting fragments by gel chromatography were performed in 80% formic acid.The amino acid sequence shows a concentration of hydrophobic amino acids in two segments of about 25 residues at the amino‐terminal and carboxy‐terminal ends. The polar residues are clustered in the middle of the polypeptide chain. The bound [14C]dicyclohexylcarbodiimide label is recovered exclusively at position 56, which is occupied by a glutamyl residue. The proteolipid from PS‐3 exhibits homology to the corresponding ATPase subunit from mitochondria. The carbodiimide‐reactive glutamyl residue occurs at the position as in the mitochondrial proteins.
A Study of Structurally Related Binding Properties of Concanavalin A Using Differential Scanning Calorimetry
Authors: BORREBAECK Carl , MATTIASSON Bo 
Published: March 2005

Differential scanning calorimetry has been used to study the interactions between concanavalin A and different carbohydrates. It was found that the binding of carbohydrate to concanavalin A stabilizes the structure of the protein as judged by an increase in transition temperature of the lectin. Furthermore, the degree of stabilization is shown to be a function of the association constant for each sugar moiety.Interactions between concanavalin A and the glycoprotein horseradish peroxidase were also studied. On changing the molar ratio of peroxidase to concanavalin A from 0.1 to 16, an initial dramatic increase in the transition temperature for concanavalin A was observed, but at ratios above four the degree of stabilization decreased; no stabilization of peroxidase was found in molar ratios above one. Results are also presented on the importance of Mn2+ and Ca2+ for the stabilization of the concanavalin A structure and for its ability to bind carbohydrates. In the presence of Mn2+ ions alone, the lectin could not form the necessary conformation for binding the carbohydrate. However, with Ca2+ ions alone the lectin was able to bind the carbohydrate ligands, as judged by the shift in its transition temperature.
Steroid Receptors and Effects of Oestradiol and Progesterone on Chick Oviduct Proteins
Authors: SUTHERLAND Robert L. , GEYNET Claudine , BINART Nadine , CATELLI Maria Grazia , SCHMELCK Paul Henry , MESTER Jan , LEBEAU Marie‐Claire , BAULIEU Etienne Emile 
Published: March 2005

After a single injection of oestradiol benzoate (1.5mg/kg) to oestrogen‐withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase α activity, adenosine‐3′,5′‐monophosphate‐dependent protein‐kinase activity and estrogen‐receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time‐course studies were performed.Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen‐receptor concentrations, a result confirmed with 11β‐methoxy‐17α‐ethynyloestradiol (R 2858), a powerful synthetic oestrogen.When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin synthesis, during the first 6–8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period.In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. Subsequently, all parameters increased, and by 18–24h after progesterone treatment, reached values higher than those observed in animals receiving oestrogen alone.