bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nonrandom Segregation of Histones during Chromatin Replication
Authors: RUSSEV George , TSANEV Roumen 
Published: March 2005

Exponentially growing Ehrlich ascites tumor cells were labelled in vivo during roughly two cell cycles by three different methods: (a) DNA was labelled during the first cell cycle with [14C]thymidine, and during the second cycle with bromodeoxyuridine + [3H]thymidine; (b) proteins were labelled during the first cell cycle with L‐[14C]valine and DNA during the second cycle with bromodeoxyuridine + [3H]thymidine; (c) both proteins and DNA were labelled during the same cell cycle with L‐(14C]valine and bromodeoxyuridine + [3H]thymidine respectively. DNA isolated from cells labelled according to method (a) was also resolved on a CsCl density gradient to show that the two successive labels are incorporated in the same DNA molecules.Chromatin was isolated by salt extraction of nuclei obtained with Nonidet P40 from Ehrlich ascites tumor cells labelled according to methods (b) and (c); 4–8% of the total protein (10–15% of the histones) were chemically bound to DNA by the procedure of Levina and Mirzabekov [Dokl. Akad. Nauk. SSSR, 221, 1222–1225 (1975)]. The unbound proteins were removed by centrifugation in a Cs2SO4/sarcosyl density gradient. The chemically linked nucleohistone was alkali‐denatured and the two DNA strands, together with the bound histones, were separated on a shallow alkaline metrizamide density gradient. The newly synthesized histones were found to be associated with the newly synthesized DNA strand while the old histones remained with the parantal DNA strand.
Mechanism of Carbamoyl‐Phosphate Synthetase
Authors: RUBIO Vicente , BRITTON Hubert Greenslade , GRISOLIA Santiago 
Published: March 2005

This paper demonstrates, by pulse‐chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its γ‐phosphoryl group to carbamoyl phosphate. This bound ATPB can react with NH3, HCO3− and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N‐acetylglutamate, but not NH3 or HCO3−, are required for this binding; the amount bound depends on the concentration of ATP (Kapp= 10–30 μM ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer.Binding of ATPB follows a slow exponential time course (t1/2 8–16 s, 22°C), independent of ATP concentration and little affected by NH3, HCO3− or by incubation of the enzyme with unlabelled ATP prior to the pulse of [γ‐32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO3− when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate (‘Cbm‐P precursor’) in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm‐P precursor when the enzyme reverts to the inaccessible conformation.Pulse‐chase experiments in the absence of NH3 and HCO3− (< 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a ‘burst’ in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO3− in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 μM) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5′)pentaphospho(5′)adenosine (Ap5A) fails to inhibit the enzyme significantly.A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321–329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent the conformational change postulated by others from steady‐state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO3−‐dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.
Anisotropy Decay of Labelled Actin
Authors: IKKAI Takamitsu , WAHL Philippe , AUCHET Jean‐Claude 
Published: March 2005

G actin, labelled presumably on cysteine‐373 with the fluorescent chromophore N‐iodoacetylN′‐(5 sulfo‐1‐naphthyl)‐ethylenediamine and purified by Sephacryl S‐200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations.It was found that these anisotropy decays could be fitted by a sum of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5°C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5°C) were introduced in order to characterize the F actin motions. The longer of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions.The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.
χ‐Carrageenase from Pseudomonas carrageenovora
Authors: McLEAN Maitland W. , WILLIAMSON Frank B. 
Published: March 2005

A χ‐carrageenase was isolated from the cell‐free medium of cultured Pseudomonas carrageenovora. From dodecylsulphate/polyacrylamide gel electrophoresis, a single protein (identified as the χ‐carrageenase) was detected in the medium. Activity against nominal carrageenan types and inspection of the products indicate the enzyme to be a χ‐carrageenase.Purification is described here by ammonium sulphate precipitation and subsequent CM‐Sepharose CL‐6B ion‐exchange chromatography. Molecular weight was estimated as 35000 by dodecylsulphate/polyacrylamide gel electrophoresis. Products of degradation were analysed by gel filtration, spectrophotometric assays and 13C nuclear magnetic resonance. These results are consistent with the product of limit digest being neocarrabiose 4‐O‐sulphate.
Norepinephrine‐Stimulated Fatty‐Acid Release and Oxygen Consumption in Isolated Hamster Brown‐Fat Cells
Authors: NEDERGAARD Jan , LINDBERG Olov 
Published: March 2005

Brown fat cells isolated from adult golden hamsters have earlier been found to respond to addition of the physiological agonist norepinephrine with an increased rate of oxygen consumption and with fatty acid release. Working with these cells, we found the following.

The presence of albumin in the incubation medium (phosphate buffer) increases norepinephrine‐induced fatty acid release and tends to stabilize the rate of oxygen consumption; bubbling of phosphate buffer with 5% CO2 in air has only a slight effect on fatty acid release.
In the presence of albumin, the norepinephrine‐induced rate of oxygen consumption is also stable in bicarbonate buffer; it is higher than in the phosphate + CO2 buffer and the brown fat cells have a higher sensitivity to norepinephrine.
20 mM phosphate (as e.g. present in a phosphate buffer) inhibits both fatty acid release and oxygen consumption.
Insulin inhibits the rate of oxygen consumption, but only at suboptimal concentrations of norepinephrine.
Atractylate inhibits submaximal norepinephrine‐induced respiration, indicating that some oxidative phosphorylation takes place in norepinephrine‐stimulated brown fat cells.
Fatty acid export from brown fat should be regarded as physiologically important.
Formate‐Nitrite Reduction in Escherichia coli K12
Authors: ABOU‐JAOUDÉ Antoine , CHIPPAUX Marc , PASCAL Marie‐Claire 
Published: March 2005

Some physiological characteristics of the formate nitrite reductase activity were studied on whole cells.

Highest level of activity was found in cells grown in anaerobiosis on glycerol in the presence of nitrite. Maximal activity was obtained at pH 8 and at 45 °C. Copper ions and cyanide inhibit the activity whereas 0.1 mM azide is without effect.
No reduction of nitrite was detected in the presence of oxygen, even at very low concentrations. Nitrate has a double effect: it competes with nitrite for electrons supplied by formate, and has an inhibitory action on nitrite reductase activity.
The stoichiometry between formate and nitrite is 3:1; the formate nitrite reduction is accompanied by CO2 evolution with the same stoichiometry.
Construction in vitro of Hybrid Plasmids Carrying All the EcoRI Fragments from λrifd18 DNA
Authors: BERNARDI Alberto , BERNARDI Françoise 
Published: March 2005

The cloning of all the eleven fragments obtained by degrading the phage λrifd18 by the restriction enzyme EcoRI into the plasmid pSF2124 has been achieved: nine of these fragments have been cloned individually, whereas two others have been cloned jointly in the same plasmid. These fragments harbor, in addition to λ genes, the genes for ribosomal proteins, the elongation Factor Tu, the β and β' subunits of RNA polymerase and the ribosomal RNAs.The clones carrying the ribosomal RNA genes have been constructed to provide convenient plasmids to determine the primary structure of ribosomal RNAs. Some further genetic manipulations in vitro have been performed on two of them to remove extraneous non‐ribosomal RNA gene sequences; the ribosomal genes purified this way have been subcloned into to the plasmid pBR322.Other clones of interest have been obtained which carry the genes for the elongation factor Tu, a number of 50‐S ribosomal proteins and the β subunit of RNA polymerase.
Light‐Induced Proton Transport by Chloroplasts Suspended in Fluid Media at Sub‐Zero Temperatures: Kinetics and Stoichiometry
Authors: OLSEN Lars F. , COX Raymond P. 
Published: March 2005

Chloroplasts suspended in a medium containing ethanediol and water (1:1, v/v) at –16°C show light‐induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics.
A 1 ms flash caused a single turnover of the photochemical centres at –16 °C. Under the same conditions 3 H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor.
The flash‐induced proton uptake was exponential and monophasic with t1/2= 3 s. The flash‐induced proton release into the thylakoid interior was biphasic, with half‐times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water.
The half‐time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half‐time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.
Modification of Peptidoglycan Structure by Penicillin Action in Cell Walls of Proteus mirabilis
Authors: MARTIN Hans Herbert , GMEINER Jobst 
Published: March 2005

Composition of cell wall peptidoglycan was studied in the gram‐negative bacterium Proteus mirabilis and in different wall‐defective growth forms of this organism which survive and multiply in the presence of penicillins. The results show that action of the classical benzylpenicillin does not proceed by a uniform mechanism of inhibition of peptide crosslinkage in peptidoglycan.Peptidoglycan of the unstable spheroplast L‐form of P. mirabilis grown in medium with 120 μg/ml benzylpenicillin contained monomers, and peptide‐crosslinked dimers and trimers of disaccharide‐peptide building blocks in a similar ratio as peptidoglycan of the normal bacterial form. Moreover, peptide side‐chains in peptidoglycan from penicillin‐grown L‐form spheroplasts were predominantly tetrapeptides l‐alanyl‐d‐γ‐glutamyl‐meso‐diaminopimelyl‐d‐alanine. Thus, specific penicillin‐sensitive enzymes of peptidoglycan synthesis, peptidoglycan transpeptidase and dd‐carboxypeptidase, must have remained uninhibited by the antibiotic in the L‐form. L‐form peptidoglycan, however, differed characteristically from peptidoglycan of normal Proteus bacteria by having a much lower content of O‐acetyl groups linked to muramic acid residues of the polymer. P. mirabilis grew actively in the form of osmotically stable spheres in medium with up to 100 μg/ml of the amidino penicillin mecillinam. Neither the peptide crosslinkage nor the O‐acetyl content of peptidoglycan were influenced to any great extent by mecillinam.Only the combined action of benzylpenicillin and mecillinam caused complete inhibition of growth in P. mirabilis, concomitant with deformation of cells to very large spheres. It is hypothesized that benzylpenicillin and mecillinam each inhibit different parts of a multiple, isofunctional enzyme system whose total inhibition requires the presence of both antibiotics.
Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 1. Medium‐Chain Fatty‐Acid Synthesis
Authors: GRUNNET Inger , KNUDSEN Jens 
Published: March 2005

Tissue slices from lactating goat‐mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long‐chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle‐free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short‐chain and long‐chain fatty acids.Terminating acyl‐thioesterases of low molecular weight could not be detected in the particle‐free supernatant.Addition of the microsomal fraction to the particle‐free supernatant induced the synthesis of medium‐chain fatty acids in proportions which were similar to those found in goat milk fat.
Localization of Phospholipids in the Membrane of Bacillus megaterium
Authors: DEMANT Erland J. F. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M. 
Published: March 2005

Phospholipase C treatment of Bacillus megaterium protoplasts at 37°C removed up to 63% of the total phospholipid without disrupting the membrane. More than 95% of the total phosphatidylethanolamine and 3′‐glucosaminylphosphatidylglycerol, 80% of the phosphatidylglycerol and about 20% of the cardiolipin and 2′‐glucosaminylphosphatidylglycerol were hydrolyzed.Similar results were obtained with isolated membranes and dispersions of lipids extracted from the membranes, demonstrating that these data do reflect, at least in part, the substrate specificity of phospholipase C and not necessarily the distribution of phospholipids over the two membrane layers.When protoplasts and isolated membranes were incubated with excess phospholipase C at temperatures in between 5°C and 25°C a different hydrolysis pattern was obtained and it was concluded that 50% of the phosphatidylethanolamine and 70% of the 3′‐glucosaminylphosphatidylglycerol are located in the outer membrane layer, the residual being in the cytoplasmic layer. At least 40% of the phosphatidylglycerol was found to be present also in the outer layer. The data do not allow conclusions about the localization of cardiolipin and 2′‐glucosaminylphosphatidylglycero1. Trinitroben‐zenesulfonic acid reacted with 50% of the phosphatidylethanolamine in protoplasts and with nearly 100% of this lipid in isolated membranes at various temperatures and concentrations, thus confirming the symmetric localization of this lipid.The data furthermore indicate that a temperature‐dependent transbilayer movement of phospholipids is induced by phospholipase C treatment of the Bacillus megaterium protoplasts.
The Phosphoenolpyruvate‐Dependent Phosphotransferase System of Staphylococcus aureus
Authors: SCHMIDT‐ADERJAN Ulrike , RÖSCH Paul , FRANK Rainer , HENGSTENBERG Wolfgang 
Published: March 2005

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine‐containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr‐56 was modified, in dinitrated HPr both Tyr‐56 and Tyr‐37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions.The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr‐A and Tyr‐B with pK values of 10.5 and 11.5 were designated Tyr‐56 and Tyr‐37 whereas Tyr‐C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr‐6 in the amino acid sequence.The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr‐56 is located at the surface, Tyr‐37 is slightly buried, Tyr‐6 is deeply buried.The nitrotyrosyl derivatives retain their biological activity.
Procaine, a Local Anesthetic Interacting with the Cell Membrane, Inhibits the Processing of Precursor Forms of Periplasmic Proteins in Escherichia coli
Authors: LAZDUNSKI Claude , BATY Daniel , PAGÈS Jean‐Marie 
Published: March 2005

Treatment of Escherichia coli cells with procaine (0.55%, w/v) results in the accumulation of precursor in addition to mature forms of two periplasmic proteins, alkaline phosphatase and glutamine‐binding protein. The precursor form of alkaline phosphatase has a higher molecular weight than the mature form by about 2600. An experimental technique is described to isolate and purify precursor forms of any presumably exported protein. After the membrane solubilization step in the presence of nonionic detergent, a peptidase is stimulated, resulting in partial cleavage of the precursors. The products of this cleavage have been identified as the mature protein and presumably the signal peptide in the case of alkaline phosphatase. The amino acid composition of this peptide, which is comprised of 25 residues, has been determined.Procaine (0.55%, w/v) causes an increase in molecular packing of lipid molecules in the membrane which might result in an alteration of membrane fluidity sufficient for selective inhibition of processing of precursors of exported proteins.
Properties of the Methane Mono‐oxygenase from Extracts of Methylosinus trichosporium OB3b and Evidence for Its Similarity to the Enzyme from Methylococcus capsulatus (Bath)
Authors: STIRLING David I. , DALTON Howard 
Published: March 2005

The methane mono‐oxygenase from Methylosinus trichosporium OB3b was soluble. The only suitable electron donor was NAD(P)H, neither sodium l‐ascorbate nor electrons derived from the oxidation of methanol could substitute for NAD(P)H. Evidence is presented for the existence of an NAD+‐linked formaldehyde dehydrogenase.
Mono‐oxygenase activity was not inhibited by a range of potential inhibitors including potassium cyanide, amytal, carbon monoxide or various metal‐chelating agents, although 8‐hydroxyquinoline and ethyne were effective in this respect.
Although the enzyme preparations were unstable on storage, the crude extract could be resolved into two components by ion‐exchange chromatography. Activity could be restored to one of the components on addition of purified components from Methylococcus capsulatus (Bath).
Cross‐reactivity of mono‐oxygenase components and other similarities between the enzymes from M. trichosporium and M. capsulatus are discussed. The properties of the M. trichosporium methane mono‐oxygenase reported here are contrasted with the properties of the same enzyme reported by others.
Permissive Effect of Dexamethasone on Glucagon Induction of Urea‐Cycle Enzymes in Perifused Primary Monolayer Cultures of Rat Hepatocytes
Authors: GEBHARDT Rolf , MECKE Dieter 
Published: March 2005

Parenchymal cells from adult rat liver, cultured in perifused monolayers, increased the levels of urea‐cycle enzymes between 15% and 60% in response to glucagon within 24 h. This stimulation was drastically enhanced by the simultaneous presence of dexamethasone, especially in the case of argininosuccinate synthetase and argininosuccinate lyase, which increased nearly threefold. Dexamethasone itself produced only negligible stimulation, but exerted a similar effect on the stimulatory action of glucagon, if it was exclusively present during 6 h prior to the glucagon treatment, suggesting a permissive action of this hormone.The effect of glucagon, particularly in the presence of dexamethasone, was mimicked by dibutyryl adenosine 3′:5′‐monophosphate, whereas epinephrine was ineffective. All stimulations induced by hormones or dibutyryl adenosine 3′: 5′‐monophosphate were abolished by cycloheximide, suggesting the involvement of protein synthesis in the induction process.Using the usual culture technique with a discontinuous supply of medium no significant effect of glucagon and dexamethasone could be measured. This striking difference between both culture systems indicates that perifusion is the more adequate in vitro system for studies of the regulation of enzyme levels. Possible reasons for the failure of hormonal stimulation of urea‐cycle enzymes in normal monolayer culture are discussed.
Phytochrome‐Induced Appearance of mRNA Activity for the Apoprotein of the Light‐Harvesting Chlorophyll a/b Protein of Barley (Hordeum vulgare)
Authors: APEL Klaus 
Published: March 2005

Illumination of dark‐grown barley plants induces a massive insertion of the light‐harvesting chlorophyll u/b protein into the developing thylakoid membrane. Light induces specifically the appearance of mRNA activity for the apoprotein of this chlorophyll‐binding protein [Apel, K. and Kloppstech, K. (1978) Eur. J. Biochem. 85, 581–588]. This change in mRNA activity can also be induced by a 15‐s red light pulse followed by 4 h darkness. The red light effect is reversed by a subsequent far‐red light treatment. It is concluded that the change in mRNA activity is controlled by phytochrome. In continuous white light the light‐harvesting chlorophyll a/b protein appears within the membrane after a lag period of 4–5 h. This lag period could be partially eliminated by a red light pulse given prior to the onset of continuous illumination. The possible role of the phyto‐ chrome‐induced appearance of mRNA activity as a rate‐limiting step of the greening process is discussed.The light‐harvesting chlorophyll a/b protein could not be detected among the plastid membrane proteins of plants which had been treated with a red light pulse alone. The assembly of the light‐harvesting chlorophyll a/b protein takes place only under continuous illumination which allows chlorophyll synthesis. Thus, the synthesis and ultimate assembly of this chlorophyll‐binding protein depends on the cooperation of two distinct light reactions.