bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exchange of Histones H1 and H5 between Chromatin Fragments
Authors: THOMAS Jean O. , REES Christine 
Published: March 2005

Histones H1 and H5 can exchange between an H1‐containing chromatin fragment from rat liver and an H1, H5‐containing fragment from chicken erythrocytes at ionic strengths from about 35 mM to 105 mM. The redistribution has reached equilibrium by ionic strength 75 mM in 1 h or less at 4°C.After exchange at ionic strength 75 mM, long fragments, whether of rat liver or chicken erythrocyte chromatin, are recovered with a higher H5:H1 ratio than short fragments, suggesting a stronger preference of H5 than of H1 for higher‐order structures which exist for long fragments at ionic strength 75 mM.Competition experiments between occupied H1 or H5 binding sites on chromatin fragments from rat liver or chicken erythrocytes and empty sites on H1‐depleted rat chromatin show that rat H1 does not distinguish between the two types of site, whereas H5 discriminates in favour of sites on native chromatin, even when the chicken fragments are too short to form higher‐order structures. (The behaviour of the chicken H1, which may be bound less tightly than rat H1, depends on the length of the chicken fragment, in a manner suggesting that fragments of 15 nucleosomes and longer can form stable higher‐order structures which have high‐affinity binding sites for both H5 and H1.)We conclude that the affinity of sites for H5 is in the order: higher‐order structures > nucleosome filament ≫ H1‐depleted chromatin. The same relative order of affinities may well apply for H1 but the discrimination is much lower. This difference between H1 and H5 seems likely to be relevant to the greater stability of H5‐containing chromatin, and in turn its transcriptional inactivity, and indeed to the mechanism of replacement of H1 by H5 during the terminal stages of erythropoiesis.
Identification of new Sialic Acids Derived from Glycoprotein of Bovine Submandibular Gland
Authors: REUTER Gerd , PFEIL Reinhard , STOLL Sabine , SCHAUER Roland , KAMERLING Johannis P. , VERSLUIS Cornelis , VLIEGENTHART Johannes F. G. 
Published: March 2005

Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N‐acetyl‐8‐O‐acetylneuraminic acid, N‐acetyl‐8,9‐di‐O‐acetylneuraminic acid, N‐acetyl‐7,8,9‐tri‐O‐acetylneuraminic acid, N‐glycoloyl‐7‐O‐acetylneuraminic acid, N‐glycoloyl‐7,9‐di‐O‐acetylneuraminic acid, N‐glycoloyl‐8,9‐di‐O‐acetylneuraminic acid, and N‐glycoloyl‐7,8,9‐tri‐O‐acetylneuraminic acid. There are also indications for the presence of N‐glycoloyl‐8‐O‐acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N‐acetylneuraminic acid, N‐acetyl‐7‐O‐acetylneuraminic acid, N‐acetyl‐9‐O‐acetylneuraminic acid, N‐acetyl‐7,9‐di‐O‐acetylneuraminic acid, N‐glycoloyl‐neuraminic acid, and N‐glycoloyl‐9‐O‐acetylneuraminic acid could be identified.Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anionexchange column (formate form) by elution with a 0–0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n‐butanol/n‐propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N‐acetylneuraminic acid, N‐acetyl‐7‐O‐acetylneuraminic acid, N‐acetyl‐9‐O‐acetylneuraminic acid, N‐glycoloylneuraminic acid, and N‐glycoloyl‐9‐O‐acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one‐dimensional and two‐dimensional thin‐layer chromatography, gas‐liquid chromatography, gas‐liquid chromatography, and gas‐liquid chromatography–mass spectrometry.
The Metabolism of Chenodeoxycholic Acid to β‐Muricholic Acid in Rat Liver
Authors: BOTHAM Kathleen M. , BOYD George S. 
Published: March 2005

The synthesis of conjugated chenodeoxycholic acid and tauro‐β‐muricholic acid in isolated rat hepatocytes was measured by radioimmunoassay. Production of tauro‐β‐muricholic acid was linear over 4 h of incubation at 37°C. The net synthesis of conjugated chenodeoxycholic acid was very much lower than that of tauro‐β‐muricholic acid.
When hepatocytes were prepared from rats in which the enterohepatic circulation had been broken, either by feeding the bile salt sequestrant, cholestyramine or by total biliary drainage for 48 h, synthesis of tauro‐β‐muricholic acid was increased compared to that in cells from control rats. Conjugated chenodeoxycholic acid accumulation during incubation of the hepatocytes was increased by cholestyramine feeding but not by total biliary drainage. These results suggest that there is a metabolic difference between the two methods of breaking the enterohepatic circulation with regard to chenodeoxycholic acid synthesis.
Hepatocytes prepared from rats given 1% cholesterol in the diet for at least 2 weeks synthesised significantly more tauro‐β‐muricholic acid than those from control rats. The total amount of conjugated cholic, chenodeoxycholic and tauro‐β‐muricholic acids synthesised by cells from cholesterol fed animals, however was not significantly different from that produced by hepatocytes from normal rats.
Exogenous taurochenodeoxycholic acid was metabolised to tauromuricholic acid by isolated hepatocytes. Production of tauro‐β‐muricholic acid reached a maximum at a concentration of 20 μM taurochenodeoxycholic acid. The total metabolism of taurochenodeoxycholic acid, however, increased linearly up to the highest concentration measured, 50 μM.
The biliary content of tauro‐β‐muricholic acid during total biliary drainage fell rapidly in the first 10 h and thereafter continued to decline, reaching a minimum after about 24 h. No significant rise was observed during the remainder of the experimental period.
It is concluded that a large proportion of the conjugated chenodeoxycholic acid synthesised by isolated hepatocytes in vitro is metabolised to tauro‐β‐muricholic acid, and therefore it is necessary to take this into account when using this system to study bile salt synthesis.
Mechanism of the Increase in Cytochrome c Oxidase Activity in Pea Cotyledons during Seed Hydration
Authors: MATSUOKA Makoto , ASAHI Tadashi 
Published: March 2005

The increase in cytochrome c oxidase activity in pea cotyledons during seed hydration was investigated with two kinds of antibodies against the enzymes purified from mitochondria of pea shoot and sweet potato root. The antibody against the pea enzyme recognized only the largest of the enzyme subunits (subunit I), while that against the sweet potato one did other three of the pea enzyme subunits: the second largest (subunit II) and the two smallest (subunits IV and V).

There was no increase in the amount of protein immunoprecipitated with the anti‐(pea enzyme) antibody in a crude fraction of cotyledon membranes during seed hydration, despite the marked increase in cytochrome c oxidase activity. Neither cycloheximide nor chloramphenicol had any effect on the activity or the amount. The crude membrane fraction from dry cotyledons contained an enzymatically inert protein precipitated with the anti‐(pea enzyme) antibody. The inert protein could be separated from the active enzyme protein through solubilization of the protein from the membranes with various concentrations of Triton X‐100. It consisted of only one kind of polypeptide with the same molecular weight as subunit I and decreased with the concomitant increase in the active enzyme protein during seed hydration.
Dry pea cotyledons also contained a soluble protein that was immunoprecipitated by antibody to the sweet potato enzyme. The soluble form of the immunoreactive protein decreased with the concomitant increase in active cytochrome c oxidase protein in the crude membrane fraction from the cotyledons during seed hydration. None of these changes during seed hydration were inhibited by cycloneximide or chloramphenicol. The soluble, immunoreactive protein contained only one kind of polypeptide, of which the molecular weight was identical to that of subunit IV.
The mitochondrial inner membranes of dry cotyledons were separated into three fractions by sucrose density gradient centrifugation. The lightest was the richest of the three in the enzymatically inert protein immunoprecipitated with the anti‐(pea enzyme)antibody; cytochrome c oxidase activity in this fraction increased immediately after the onset of seed hydration.These results indicate that a membrane‐bound, free form of subunit I and a soluble, free form of subunit IV of cytochrome c oxidase exist in dry pea cotyledons. We propose that these free forms of subunits are assembled with the other subunits to form the active cytochrome c oxidase during seed hydration.
Reaction of Monosubstituted Hydrazines and Diazenes with Rat‐Liver Cytochrome P450
Authors: BATTIONI Pierrette , MAHY Jean‐Pierre , DELAFORGE Marcel , MANSUY Daniel 
Published: March 2005

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Soret peak at 446 nm. Upon oxidation of the [cytochrome P450‐Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex charaterized by a Soret peak at 486 nm is formed. The latter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450‐Fe(III) or in situ oxidation of methylhdrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO.Reactions of ethyldiazene or benzyldiazene with cytochrome P450‐Fe(III) afforded similar complexes characterized by Soret peaks around 480 nm.These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN = NH with hemoglobin and iron‐porphyrins, are consistent with a [cytochrome P450‐Fe(II)(RN = NH)] structure for the 446‐nm‐absorbing complexes and a σ‐alkyl cytochrome P450‐Fe(III)‐R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a σ‐cytochrome P450‐Fe(III)‐Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450‐Fe(III)‐R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines.
Utilization of Energy Stored in the Form of Na+ and K+ Ion Gradients by Bacterial Cells
Authors: BROWN Igor I. , GALPERIN Michael Yu. , GLAGOLEV Alexey N. , SKULACHEV Vladimir P. 
Published: March 2005

The hypothesis that Na+ and K+ gradients have an energy storing function [V. P. Skulachev (1978) FEBS Lett. 87, 171–176] has been tested in experiments with Escherichia coli, the marine bacterium Vibrio harveyi, an extremely halophilic Halobacterium halobium and a fresh‐water cyanobacterium Phormidium uncinatum from Lake Baikal living at an extermely low salt concentration. The capability of these microorganisms to maintain was compared using motility as a ‐supported function. It was found that in all cases the gradient of monovalent cations is competent to prolong the period of active motility after other energy sources are exhausted. Maximal prolongation was found in H. halobium, which in a Na+ medium was still motile when light was switched off for 9 h under anaerobic conditions. In V. harveyi the motility was maintained for 1 h, in E. coli for about 10 min and in Ph. uncinatum for about 2 min. Thus the buffer capacity of the monovalent cation gradient is proportional to the content of these cations in the habitat. It was also found that in Ph. uncinatum only ΔpK is effective, whereas in E. coli and V. harveyi both ΔpK and ΔpNa are.In E. coli when the K+ release is completed and the cells become motionless, motility can be temporarily restored by adding NaCl which initiates an H+ efflux.Under conditions of exhaustion of energy sources, the Na+ and K+ gradient was shown to stabilize potential in H. halobium cells, measured with a tetraphenylphosphonium probe.In H. halobium and E. coli, the anaerobic ATP level was found to stabilize when the Na+ and K+ gradients were present. Addition of N, N′‐dicyclohexylcarbodiimide destabilized this level, which indicated that Na+ and K+ gradients could support de novo ATP synthesis.It is concluded that the data obtained are in agreement with the concept of the energy storing by the Na+ and K+ gradients. Other functions of these gradients and the mechanisms of their formation are discussed.
Kinetic Evidence for an Activation Step Following Binding of Human Interferonα2 to the Membrane Receptors of Daudi Cells
Authors: MOGENSEN Knud E. , BANDU Marie‐Thérèse 
Published: March 2005

A single species of human interferon α (IFN] was labelled with 125I to high incorporation for binding studies on the B‐lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination.Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same intial culture of cells. 125I‐labelled IFNα (IFNα prepared from Escherichia coli carrying human α2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 ± 0.07 days−1) at a mean concentration of 235000 ± 20000 cells ml−1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibirum some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the ‘steady‐state’, appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occuring consecutively over the wholw of the dose range studied (1 – 100 U/ml; 0.25–25pM IFN).In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double‐reciprocal plot (i.e. in analogy to Michaelis‐Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 PM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited.We propose that the first reaction represents binding of IFN to surfac receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.
Regulation of Ca2+Efflux in Rat Liver Mitochondria
Authors: BERNARDI Paolo , AZZONE Giovanni Felice 
Published: March 2005

The paper analyzes the relationship between membrane potential (ΔΨ), steady state pCa0(‐log [Ca2+] in the outer aqueous phase) and rate of ruthenium‐red‐induced Ca2+efflux in liver mitochondria. Energized liver mitochondria maintain a pCa0of about 6.0 in the presence of 1.5 mM Mg2+and 0.5 mM Pi. A slight depression ofΔΨresults in net Ca2+uptake leading to an increased steady state pCa0. On the other hand, a more marked depression ofΔΨresults in net Ca2+efflux, leading to a decreased steady‐state pCa0. These results reflect a biphasic relationship betweenΔΨand pCa0, in that pCa0increases ofΔΨup to a value of about 130 mV, whereas a further increase ofΔΨabove 130 mV results in a decrease of pCa0. The phenomenon of Ca2+uptake following a depression ofΔΨis independent of the tool used to affectΔΨwhether by inward K+current via valinomycin, or by inward H+current current through protonophores or through, F1‐ATP synthase, or by restriction of e−flow.The pathway for Ca2+efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCa0at steady state and by an increased rate of ruthenium‐red‐induced Ca2+efflux. By restricting the rate of e−flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium‐red‐induced Ca2+efflux on the value ofΔΨis observed, in that the rate of Ca2+efflux increases with the value ofΔΨ. The pCa0is linearly related to the rate of Ca2+efflux.Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP‐supplemented mitochondria, is followed by a phase of Ca2+uptake, which is reversed by atractyloside.These findings support the view that Ca2+efflux in steady state mitochondria occurs through an independent,ΔΨ‐controlled pathway and that changes ofΔΨduring oxidative phosphorylation can effectively modulate mitochondrial Ca2+distribution by inhibiting or activating theΔΨ‐controlled Ca2+efflux pathway.
The Nature of the Electron Spin Resonance Signal during Aerobic Uptake of Mn2+ in Mitochondria from Rat Liver
Authors: BRAGADIN Marco , POZZAN Tullio , AZZONE Giovanni Felice 
Published: March 2005

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high‐energy membrane sites or to complexes of Mn2+ with inorganic phosphate.Identification of the spin exchange signal with a Mn‐Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N‐ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Piis generated in the matrix, in the presence of N‐ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal.The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50–60 nmol × mg protein −1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations.The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93–99] that the spin exchange singal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.
Antigenic determinants of human placental and testicular placental‐like alkaline phosphatases as mapped by monoclonal antibodies
Authors: MILLÁN José Luis , STIGBRAND Torgny 
Published: March 2005

Placental alkaline phosphatase (PLAP) in humans shows a high degree of genetic polymorphism as disclosed by electrophoretic analysis. Human testes contain trace amounts of a PLAP‐like enzyme, that although immunologically cross‐reactive with PLAP, shows unique catalytic properties.As an alternative approach to study enzyme polymorphism we have developed monoclonal antibodies to purified allelic variants of PLAP. Five different monoclonal antibodies are described in this report. The antibodies react with different epitopes on the PLAP molecule. Both conformational dependent and independent determinants are detected. Two epitopes are modified when comparing the S and F allelic variants of PLAP. One epitope is common to PLAP and the intestinal isoenzyme of alkaline phosphatase. The five epitopes appear to be mapped on two rather distant antigenic domains. Combinations of any two antibodies binding to different domains give immunoprecipitates with PLAP on Ouchterlony tests and give good response in sandwich enzyme‐linked immunosorbent assays.A study of PLAP‐like enzyme in 32 individual testis samples indicates differences in four of the epitopes when compared with PLAP. Four types of testicular enzymes can be distinguished based on their reactivities. These results indicate structural differences between the testicular PLAP‐like enzyme and PLAP. These differences are compatible with an underlying genetic mechanism.
Localization of the tight ADP‐binding site on the membrane‐bound chloroplast coupling factor one
Authors: CZARNECKI Joseph J. , ABBOTT Marilyn S. , SELMAN Bruce R. 
Published: March 2005

The photoaffinity analog 2‐azido‐ADP (2‐azidoadenosine 5′‐diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2‐azido‐ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2‐azido‐ADP competitively inhibited ADP tight binding (Ki= 1.4 μM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 μM) was similar to that observed for ADP (2 μM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re‐energization; (d) the tight binding of 2‐azido‐[β‐32P]ADP was completely prevented by ADP; (e) the analog inhibited the light‐triggered ATPase activity at micromolar concentrations.Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2‐azido‐[β‐32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the β subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the β subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.
Purification of stalk‐cell‐inducing morphogens from Dictyostelium discoideum
Authors: KAY Robert R. , DHOKIA Babu , JERMYN Keith A. 
Published: March 2005

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low‐Mr factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262, 717–719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76, 308–312]. These differentiation‐inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD‐2 resin, extraction into hexane and two steps of reverse‐phase high‐pressure liquid chromatography (HPLC). Our results show the following.

HPLC resolves a major stalk‐cell‐inducing activity (DIF‐1) and at least four minor and more polar activities (DIFs 2–5).
DIF‐1 has been purified at least 3000‐fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF‐1 can be explained in part by the loss of non‐specific stimulatory (‘helper’) factors during the purification. A few micrograms purified DIF‐1 were obtained from 1012 cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM.
The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF‐5 was partially sensitive and DIF‐4 resistant. Other properties of DIF‐1 suggest that it is a non‐polar molecule of Mr < 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties.
The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.