bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutron‐Scattering Studies of Accurately Reconstituted Nucleosome Core particles and the Effect of Ionic Strength on Core Particle Structure
Authors: SIBBET Gray J. , CARPENTER Brian G. , IBEL Konrad , MAY Roland P. , KNEALE G. Geoffrey , BRADBURY E. Morton , BALDWIN John P. 
Published: March 2005

Chicken erythrocyte nucleosome core particles can be dissociated quantitatively into histone (H3, H4)2 bound to 146 base pairs of DNA, and 2(H2A, H2B). Reconstitution of core particles from the two components produces an 85% yield of particles which neutron scattering studies show to be accurate stoichiometrically and indistinguishable from native core particles: the radii of gyration of the shape, the protein components and the DNA components of the particles are 4.02 nm, 3.3 nm and 4.95 nm respectively. The largest distance and most probable distance which can be drawn in the particles are 11.5 nm and 4.3 nm respectively. The molecular weight of the particles is identical to that of control ‘native’ core particles. All of these values, within limits of error, are the same as known values for ‘native’ core particles. These experiments confirm the essential role of histones H3 and H4 in the initial organisation of core‐particle structure, make possible the manufacture of perfectly pure and homogeneous core‐particle preparations and allow the 100% incorporation of labelled or modified histones.Neutron scattering studies of core particles at high contrats (in D2O and H2O) have been carried out over a range of ionic strengths and pH. No change in structure is detected down to pH 5.5 in 20 mM NaCl or down to ionic strength 2.0 mM at pH 7.
Expression of Interferon‐α and Interferon‐β Genes in Human Lymphoblastoid (Namalwa) Cells
Authors: SHUTTLEWORTH John , MORSER John , BURKE Derek C. 
Published: March 2005

Treatment of human lymphoblastoid (Namalwa) cells with Sendai virus induced the coordinate synthesis of both IFN‐α and IFN‐β interferon mRNAs. One sub‐line of Namalwa cells (WRL) produced no IFN‐β activity, although IFN‐β mRNA was induced and was associated with polysomes. The IFN‐α mRNA was heterogeneous, ranging in size over 1.20–1.35 × 103bases, probably because of variation in the size of the transcribale DNA in the α‐gene family. The IFN‐β mRNA was monodisperse with a size of 1.05 × 103bases. The kinetics of accumulation and decay of both IFN‐α and IFN‐β mRNAs, as assessed by hybridization with cDNA probes, were very similar to those of translatable interferon mRNA, as assessed by translation inXenopusoocytes.Treatment of the cells with butyrate or 5′‐bromodeoxyuridine increased the amount of hybridizable IFN‐α and IFN‐β mRNA about 15‐fold and 4‐fold respectively, again demonstrating coordinate control of IFN‐α and IFN‐β production.
Characterisation of a Reconstituted Mg‐ATP‐Dependent Protein Phosphatase
Authors: RESINK Therese J. , HEMMINGS Brian A. , TUNG H. Y. Lim , COHEN Philip 
Published: March 2005

Homogenous preparations of the catalytic subunit of protein phosphatase‐1 and inhibitor‐2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg‐ATP‐dependent protein phosphatase in that preincubation with glycogen synthase kinase‐3 and Mg‐ATP is required to generate activity. Activation results from the phosphorylation of inhibitor‐2. The molar concentrations of protein phosphatase‐1 and inhibitor‐2 in rabbit skeletal muscle (0.25–0.5 μM) are similar.Incubation of the reconstituted Mg‐ATP‐dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor‐2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase‐3 and Mg‐ATP provided that inhibitor‐2 is added. This exogenous inhibitor‐2 appears to displace the fragments of inhibitor‐2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S. D., Vandenheede, J. R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231–10234] that inhibitor‐2 can function as an ‘activator’ as well as an inhibitor of the Mg‐ATP‐dependent protein phosphatase.Incubation of the catalytic subunit of protein phosphatase‐1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive from that could be partially reactivated by manganese ions, but not by glycogen synthase kinase‐3 and Mg‐ATP. Conversely, the reconstituted Mg‐ATP‐dependent protein phosphatase could only be activated by glycogen synthase kinase‐3 and Mg‐ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase‐1 to a manganese‐ion dependent form is a quite separate phenomenon from the formation of the Mg‐ATP‐dependent protein phosphatase.Inhibitor‐2 can inactivate protein phosphatase‐1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase‐3 and Mg‐ATP. This occurs at higher concentrations of inhibitor‐2 than those required to form the Mg‐ATP‐dependent protein phosphatase, and appears to result from the binding of inhibitor‐2 to a distinct site on the enzyme.
On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by 13C‐Nuclear‐Magnetic‐Resonance Relaxation
Authors: MOONEN Chrit T. W. , MÜLLER Franz 
Published: March 2005

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non‐protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (>90%13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole‐dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein‐bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field‐dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP.In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.
Sequence Requirement for Transcription in vitro of the Human Corticotropin/β‐Lipotropin Precursor Gene
Authors: NOTAKE Mitsue , KUROSAKI Tomohiro , YAMAMOTO Tokuo , HANDA Hiroshi , MISHINA Masayoshi , NUMA Shosaku 
Published: March 2005

Using HeLa whole cell extracts, we have demonstrated that transciption in vitro of the cloned human and bovine corticotropin/β‐lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5′‐deletion mutants of a fusion gene that contains the 5′‐flanking sequence and capping site of the human corticotropin/β‐lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the ‘TATA box’ present in this region seems to be the main promoter element for transcription of the human corticotropin/β‐lipotropin precursor gene in the HeLa cell‐free system.We have also developed a transcription system in vitro from the corticotropin‐producing mouse pituitary tumor cell line AtT‐20 in culture. Deletion mapping of the fusion gene promoter has indicated that the ‘TATA box’ region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT‐20 cell‐free system, but hardly in the HeLa cell‐free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT‐20 cell extract is responsible for the modulation of transcription of the human corticotropin/β‐lipotropin precursor gene.
Molecular Forms of β‐N‐Acetylhexosaminidase in Epstein‐Barr Virus‐Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Tay‐Sachs Disease
Authors: SALVAYRE Robert , MARET Arlette , NEGRE Anne , LENOIR Gilbert , VUILLAUME Michèle , ICART Josette , DIDIER Jacqueline , DOUSTE‐BLAZY Louis 
Published: March 2005

In whole leukocytes and in lymphocytes from normal subjects, the percentage activity of heat‐stable β‐N‐acetylhexosaminidase (30 ± 5% and 45 ± 5%, respectively) was higher than in the transformed lymphoid cell line (19 ± 3%). In Tay‐Sachs transformed cells as well as non‐transformed lymphocytes, β‐N‐acetylhexosaminidase was almost completely heat‐stable (95–98%).
In the transformed cells from normal subjects, the β‐N‐acetylhexosaminidase B (Hex B) activity (5% of total) was significantly lower than in blood lymphocytes (average 25–30% of total activity), whereas Hex A and Hex I were similar in the either cell type.
Blood lymphocytes and lymphoid cell lines established from a Tay‐Sachs patient lacked heat‐labile Hex A and expressed high heat‐stable Hex I and Hex B activities (3–6‐fold).
After neuraminidase treatment, Hex A peak sharpened while Hex I peaks switched to higher pI than normal Hex I, in the region of Hex B. PreHex A/S pI was not affected.
Hydrolytic properties using the both substrates (4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside and 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranoside) of each molecular form were similar in transformed and non‐transformed cells. Data derived from the use of a mixture of substrates were consistent with the model which proposes a common active site for either substrate in the case of preHex A, Hex B and Hex I, but not for Hex A.Thus Epstein‐Barr virus‐transformed lymphoid cell lines represent an accurate model system for studies on Tay‐Sachs disease.
Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDEN Bertil , PETTERSSON Gösta 
Published: March 2005

Aldehyde binding to liver alcohol dehydrogenase in the absence and presence of coenzymes has been characterized by spectrometric equilibrium methods, using auramine O and bipyridine as reporter ligands.
Free enzyme shows a significant affinity for aldehydes, and equilibrium constants for dissociation of the binary complexes formed with typical aldehyde substrates are reported. Binary‐complex formation does not lead to any detectable inner‐sphere coordination of aldehydes to the catalytic zinc ion of the enzyme subunit.
Complex formation with NAD+ or NADH increases the affinity of the enzyme for aromatic aldehydes by a factor of 1.8–3.5 and 6–17, respectively. Benzaldehyde and dimethylaminocinnamaldehyde binding to the enzyme · NAD+ complex is not detectably associated with inner‐sphere coordination of the aldehyde to zinc.
It is concluded that binding of NADH is required to induce catalytically adequate bonding interactions between enzyme and aromatic aldehydes. The effect of reduced coenzyme in this respect is attributed to hydrophobic interactions leading to dehydration of the active‐site region, which allows aldehyde substrates to compete successfully with water for inner‐sphere coordination to the catalytic zinc ion. Oxidized coenzyme is proposed to have a similar promoting effect on metal coordination of aldehydes which function as substrates for the dismutase activity of the enzyme.
The Interaction of Mammalian Medium‐Chain Hydrolase with Yeast Fatty Acid Synthetase
Authors: SLABAS Antoni R. , ORMESHER Jane , ROBERTS Peter A. , SIDEBOTTOM Christopher M. , TOMBS Michael P. , JEFFCOAT Roger , JAMES Anthony T. 
Published: March 2005

The interaction of rat mammary gland medium‐chain thioesterase with yeast fatty acid synthetase has been investigated.

Medium‐chain thioesterase interacts with yeast fatty acid synthetase causing premature chain termination of the fatty acids synthesized from acetyl‐CoA and malonyl‐CoA. This effect is most marked under conditions of rate‐limiting malonyl‐CoA availability.
Immobilized yeast fatty acid synthetase specifically binds rat mammary gland medium‐chain thioesterase. This interaction has been used to purify medium‐chain thioesterase to near homogeneity from samples of rat mammary gland cytosol.
The stoichiometry of binding of medium‐chain thioesterase to yeast fatty acid synthetase has been investigated. Yeast fatty acid synthetase binds 5.7 ± 1 mol medium‐chain thioesterase/mol yeast fatty acid synthetase.
It is concluded that yeast fatty acid synthetase has a medium‐chain thioesterase binding site.
Ganglioside Biosynthesis in Golgi Apparatus of Rat Liver
Authors: YUSUF Harun K. M. , POHLENTZ Gottfried , SCHWARZMANN Günter , SANDHOFF Konrad 
Published: March 2005

Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3 : CMP‐NeuAc sialyltransferase, GD3 synthase; GM3 : UDP‐GalNAc galactosaminyltransferase, GM2 synthase) were 50–60‐times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6‐fold and 20‐fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50μg Golgi protein and 1 nmol UDP‐GalNAc, optimal stimulation of GM2 synthase was obtained with 20 μg of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceds (by about 40%) that obtained with optimal amount (200μg) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or Mn2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis.Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 μg tunicamycin and more than 85% by 10 μg tunicamycin, per 50 μg Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 μg of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. All these results indicate tht phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier‐mediated transport, e. g. of nucleotide sugars, across Golgi vesicles.
Na+‐Dependent Uptake of 4‐Azidophenylalanine by Pig Intestinal Microvillus Vesicles
Authors: NORÉN Ove , SJÖSTRÖM Hans , LARSEN Per 
Published: March 2005

A procedure for the synthesis of tritiated 4‐azidophenylalanine in a radioactively homogeneous form is described. The characteristics of the 4‐azidophenylalanine uptake by pig intestinal microvillar vesicles and its interaction with the neutral amino acid transport have been studied. 4‐Azidophenylalanine is transported into an osmotically sensitive space and the transport is sodium dependent. It is demonstrated that 4‐azidophenylalanine transport is inhibited by l‐alanine and l‐phenylalanine but not by l‐lysine and d‐glucose. Likewise 4‐azidophenylalanine inhibited the initial transport of l‐lysine and d‐glucose was unaffected. Treatment of microvillar vesicles with 4‐azidophenylalanine in combination with photolysis irreversibly inactivated the l‐alanine transport.Photolabelling in the presence of sodium using tritiated 4‐azidophenylalanine resulted in labelling of 10 peptides, all found in the fraction insoluble in Triton X‐100. Omission of sodium from the reaction mixture or addition of l‐alanine did not specifically abolish labelling in any of the labelled components.
The Influence of Cholesterol Incorporation and Removal on Lipid‐Bilayer Viscosity and Electron Transfer in Rat‐Liver Microsomes
Authors: ARCHAKOV Alexander I. , BORODIN Evgeni A. , DOBRETSOV Gennadi E. , KARASEVICH Elena I. , KARYAKIN Alexander V. 
Published: March 2005

The incorporation and removal of cholesterol from rat liver microsomes was used as a methodical approach to investigate the molecular organization of microsomal redox‐chains.It was shown that the incorporation of cholesterol in microsomes increases and removal of cholesterol decreases lipid bilayer viscosity as indicated from the rate of fluorescent probe‐pyrene eximerisation in cholesterol‐enriched and cholesterol‐depleted microsomes.The increase of membrane viscosity slows down the initial rates and decreases the rate constants of cytochrome b5 reduction by NAD(P)H, whereas the decrease of membrane viscosity enhances the initial rates and increases the rate constants of these reactions. The rates of cytochrome P450 reduction by reduced pyridine nucleotides do not depend on the viscosity of lipid bilayer. The incorporation and removal of cholesterol from microsomes was not followed by any essential changes in the rates of dimethylaniline N‐demethylation, aniline p‐hydroxylation, p‐nitroanisole O‐demethylation, oxygen consumption, oxidation of NADH and NADPH.Thus the reduction of cytochrome b5 by NADH and NADPH is the diffusion‐dependent reaction in the redoxchains of microsomes only.
The Properties of Citrate Transport in Membrane Vesicles from Bacillus subtilis
Authors: BERGSMA Jack , KONINGS Wil N. 
Published: March 2005

The uptake system for citrate is induced in Bacillus subtilis W23 by growth in the presence of citrate and only membrane vesicles isolated from these cells show energy‐dependent citrate uptake. Citrate transport in membrane vesicles is strictly dependent on the presence of divalent cations such as Mg2+, Mn2+, Zn2+, Ba2+, Be2+, Ca2+, Cu2+, Co2+, or Ni2+. The initial rate of citrate transport increases with the divalent cation concentration up to a maximum. The maximum initial rate of citrate uptake is reached with 2 mM Mg2+.The cations form stable chelates with citrate. The metal citrate complex is the transported solute. This is demonstrated for citrate uptake in the presence of Ca2+. Membrane vesicles from citrate‐grown cells accumulate Ca2+ and citrate only if both solutes are present. Citrate and Ca2+ are accumulated in equimolar quantities. The uptake of Ca2+ but not of citrate is inhibited by Mg2+.Uptake of the metal‐citrate complex is inhibited by the uncoupler carbonylcyanide p‐trifluoromethoxyphenyl‐hydrazone and in the presence of K+ ions by valinomycin and nigericin. The inhibitory effects correlate with the effects observed on the components of the proton‐motive force, indicating that the proton‐motive force is a driving force for metal‐citrate transport. The number of protons (n) symported with the metal‐citrate complex has been determined under different experimental conditions from the steady state levels of citrate accumulation, the electrical potential and pH gradient. This number varies from 1 at pH 4.7 to 2 at pH 8.0.
Comparative Binding of Ethidium and Three Azido Analogs to Dinucleotides: Affinity and Intercalation Geometry
Authors: LAUGAA Philippe , DELBARRE Alain , LE PECQ Jean‐Bernard , ROQUES Bernard P. 
Published: March 2005

Geometrical and thermodynamic information has been obtained from theoretical analysis of both visible and 1H‐NMR spectroscopic binding isotherms of ethidium and three photoactivable derivatives (8‐azido‐ethidium, 3‐azido‐ethidium and 3,8‐diazido‐ethidium) to self‐complementary ribodinucleosides. The following results have been obtained.

Interaction with pyrimidine(3–5′)purine sequences is well accounted for by multicomponent equilibria involving self‐association of the dyes in oligomers, formation of 1:1 and 2:1 (nucleoside:dye) complexes. This model provided evidence for intercalation of all dyes, though with weaker affinity in the case of diazido‐ethidium (2 × 106 M−2 vs 6 × 107 M−2). Moreover 3‐azido‐ethidium was shown to intercalate into cytidylyl(3′–5′)guanosine (CpG) with its phenyl group lying in the major groove of the minihelix. This geometry is inverted with respect to that of all other compounds. It should be emphasized that visible and 1H‐NMR techniques independently provided similar results (intercalation, affinity constants) therefore supporting this stepwise model.
Interaction of all dyes with purine(3′–5′)pyrimidine sequences is not intercalative, even at low temperature (4°C), but is well described by self‐association of the dyes and formation of 1:1 (nucleoside:dye) complexes.Regarding the reversible DNA intercalation process, these studies show that 8‐azido‐ethidium is the only photoactivatable derivative which behaves exactly as ethidium. Therefore 8‐azido‐ethidium can be used as a covalent probe to investigate the DNA‐related cytotoxic effects of ethidium.
The Lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505
Authors: TAHARA Yasutaka , WILKINSON Stephen G. 
Published: March 2005

Structural studies have been carried out on the O‐specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The O‐specific polysaccharide has a tetrasaccharide repeating‐unit containing residues of l‐rhamnose (Rha), 2‐acetamido‐2‐deoxy‐d‐glucose (GlcNAc), 2‐acetamido‐2‐deoxy‐l‐galacturonic acid (GalNAcA), and 2,4‐diacetamido‐2,4,6‐trideoxy‐d‐glucose (BacNAc2). The following structure has been assigned to the repeating‐unit:→3)Rhap(β1→6)GlcpNAc(α1→4) GalpNAcA(α1→3)BacpNAc2(α1→.The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3‐deoxyoctulosonic acid, and amide‐bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3:1:1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6‐substituted.