bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Overall Partitioning of Anthracyclines into Phosphatidyl‐Containing Model Membranes Depends Neither on the Drug Charge Nor the Presence of Anionic Phospholipids
Authors: Gallois Laurence , Fiallo Marina , Laigle Alain , Priebe Waldemar , Garnier‐Suillerot Arlette 
Published: July 2004

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P‐glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P‐glycoprotein‐mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point.The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions.Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles.The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3′‐hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13‐hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol.We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.
Selenium‐Dependent Peroxidases Suppress 5‐Lipoxygenase Activity in B‐Lymphocytes and Immature Myeloid Cells
Authors: Werz Oliver , Steinhilber Dieter 
Published: July 2004

Differentiation of HL‐60 cells by dimethylsulfoxide induces 5‐lipoxygenase protein expression, but only low cellular 5‐lipoxygenase activity. Similarly, B‐lymphocytes express 5‐lipoxygenase protein and show activity in cell homogenates but not in intact cells. Here, we demonstrate that suppression of cellular 5‐lipoxygenase activity in these cell lines is serum dependent and that the serum effect can be mimicked by selenium. Selenium‐dependent inhibition of 5‐lipoxygenase activity was also observed in the corresponding cell homogenates or 100000×g supernatants when dithiothreitol or glutathione (GSH) was added. The properties of the endogenous selenium‐dependent inhibitor, i.e. molecular mass, utilization of GSH and dithiothreitol as substrates, sensitivity to iodacetate, inhibition of 5‐lipoxygenase activity in the presence of the GPx‐1 inhibitor mercaptosuccinate, suggest that a selenoenzyme with properties of the phospholipid hydroperoxide glutathione peroxidase (GPx‐4) is responsible for the 5‐lipoxygenase inhibition in BL41‐E95‐A and immature HL‐60 cells.Differentiation of HL‐60 cells in the presence of 1,25‐dihydroxyvitamin D3 and transforming growth factor‐β(TGFβ) upregulated cellular 5‐lipoxygenase activity regardless of whether the cells were grown with or without serum or selenium. Also, 5‐lipoxygenase activity in homogenates or 100000×g supernatants of 1,25‐dihydroxyvitamin D3/TGFβ differentiated HL‐60 cells and of human granulocytes was not inhibited by dithiothreitol or GSH. Thus, after 1,25‐dihydroxyvitamin D3/TGFβ differentiation, HL‐60 cells resemble normal granulocytes with respect to the high 5‐lipoxygenase activity in intact cells and to the dithiothreitol effects in broken cell preparations. Combination experiments with 100000×g supernatants of BL41‐E95‐A cells and neutrophils revealed that the high 5‐lipoxygenase activity of granulocytes is due to stability of the 5‐lipoxygenase catalytic activity against selenium‐dependent peroxidases, but not to low peroxidase activity. Our data suggest that the capability of mature myeloid cells to release large amounts of leukotrienes after stimulation is due to a peroxidase‐insensitive 5‐lipoxygenase catalytic activity.
Evaluation of the Secondary Structure of Vaccinia‐Virus Thymidine Kinase by Circular‐Dichroism Spectroscopy of Overlapping Synthetic Peptides
Authors: Behrends Henrik W. , Beck‐Sickinger Annette G. , Folkers Gerd 
Published: July 2004

A new approach is reported that includes multiple‐peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia‐virus thymidine kinase (TK). We divided the sequence of the vaccinia‐virus TK into 82 peptides of IS residues that overlapped by 13 residues and covered the complete sequence of vaccinia‐virus TK. All peptides were synthesized by solid‐phase multiple‐peptide synthesis by means of the Fmoc/tert ‐butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary‐structure evaluation led to determination of a vaccinia‐virus‐TK secondary‐structure pattern.Consecutive peptides with α‐helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of α‐helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear β‐sheet conformation, or CD spectra of mixed secondary‐structure content were observed for β‐sheets. Therefore, the exact localization of β‐sheet‐containing residues was deduced by comparison with isofunctional sequence‐dissimilar proteins. We identified seven α‐helices and six β‐sheet‐containing regions, which we used for a secondary‐structure model of the vacciniavirus TK protein.
Analysis of Nucleotide Binding by a Vacuolar Proton‐Translocating Adenosine Triphosphatase
Authors: Webster Lorna C. , Apps David K. 
Published: July 2004

The vacuolar‐type proton‐translocatine adenosine triphosphatase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The binding of nucleotides to the enzyme was studied by quantifying their effects on the rate of inactivation by N‐ethylmaleimide (MalNEt) of ATP‐dependent proton translocation, and by direct measurement of the binding of [3H]MgADP. The results of these experiments are consistent with a model of the enzyme that had been developed as a result of kinetic experiments, the features of which are that the enzyme exists in two states, each containing three nucleotide‐binding sites on catalytic subunits, and that nucleoside diphosphates regulate the enzyme by binding with high affinity to a single site in the inactive T state of the enzyme.Under the conditions of the experiments, MalNEt inactivated the ATPase in a pseudo‐first order reaction. Rate constants of inactivation were reduced in the presence of MgADP, MgIDP and free ADP; the kinetics of protection suggested that the two conformational states of the enzyme were inactivated at different rates and also confirmed the existence of two different types of binding site for MgADP. Low nucleotide concentrations afforded partial protection from MalNEt; this was ascribed to binding of nucleotide to the regulatory site causing a shift in the conformational equilibrium towards the T state, which was more slowly inactivated than the unliganded R state of the enzyme. At higher nucleotide concentrations, binding at the catalytic site afforded complete protection from MalNEt. Protection by MgADP[S] and magnesium 2′‐ and 3′‐O ‐[4‐benzoylbenzoyl]adenosine 5′‐triphosphate showed simpler kinetics but was also consistent with previously reported kinetic results.Analysis of subunit labelling with [3H]MalNEt showed that the three 72‐kDa (catalytic) subunits were alkylated by MalNEt with similar rate constants, consistent with a symmetrical arrangement of the catalytic subunits, in contrast to the situation in F‐type ATPases.Analysis of the binding of [3H]MgADP also confirmed the results of kinetic experiments. MgADP was shown to bind to the enzyme with an apparent dissociation constant of about 66 nM; assuming that the nucleotide binds only to the T‐state, the true dissociation constant is < 1 nM. Using Blue Native polyacrylamide gel electrophoresis to separate the holo‐ATPase from the membrane sector, the stoichiometry of binding was calculated to be 0.6 mol/mol enzyme, confirming the existence of a single regulatory site for MgADP. However, binding of MgADP to the enzyme was much slower than could be accounted for by the measured dissociation constants, suggesting that it is rate limited by a step such as a protein conformational change. Treatment designed to remove endogenous nucleotide had no effect on the rate or extent of binding of MgADP.
The Osmotic Properties and Free Energy of Formation of the Actomyosin Rigor Complexes from Rabbit Muscle
Authors: Magri Ermes , Cuneo Paola , Trombetta Giorgio , Grazi Enrico 
Published: July 2004

We have studied the osmotic properties of the calcium‐regulated actomyosin complexes from skeletal muscle at the protein osmotic pressure of 18 kPa and at different actin‐to‐myosin molar ratios. Essentially, protein solutions were equilibrated against a solution of poly(ethylene glycol) 40000 of known macromolecular osmotic pressure. At the end of the equilibration the water and the protein masses of the protein solutions were determined gravimetrically and the protein molar concentration was calculated.In this reconstructed system we have found following, at the actin‐to‐molar ratio of 2.6 (the most likely stoichiometry of these two proteins in the dense region of the A band) the average distance between the myosin filaments is 34.2 nm, this equals the interfilament distance in the intact fibre of muscle in rigor, at the sarcomere length of 3.38 μm. The formation of the F‐actin‐myosin and of the tropomyosin–F–actin‐myosin rigor complexes involves the largest free energy changes, ‐5.38 kJ/mol myosin and ‐5.67 kJ/mol myosin, respectively.The formation of the troponin–tropomyosin–F‐actin–myosin(Ca) rigor complex from myosin and troponin–tropomyosin–F‐actin(Ca) occurs with the free energy change of ‐3.43 kJ/mol myosin. Of these ‐3.43 kJ, ‐1.81 kJ are provided by the endergonic conversion of troponin–tropomyosin–F‐actin(EGTA) into troponin–tropomyosin–F‐actin(Ca). The transition of myosin and of troponin–tropomyosin–F‐actin(EGT.4) into the ‐F‐actin‐myosin(Ca) rigor complex is accompanied by a 5.8% increase of volume. The increase of volume is due to a large influx of water, which is essentially protein‐hydration water.
The Mechanism of Proline/Glutamate Antiport in Rat Kidney Mitochondria
Authors: Atlante Anna , Passarella Salvatore , Pierro Pasqualina , Di Martino Catello , Quagliariello Ernesto 
Published: July 2004

Proline/glutamate antiport in rat kidney mitochondria has been studied in terms of two different features: energy dependence and glutamate‐carrier contribution to accomplish proline movement across the mitochondrial membrane.Energy dependence of the proline/glutamate antiporter in rat kidney mitochondria has been investigated by means of both spectroscopic measurements and isotopic techniques, using either normal or [14C]glutamate‐loaded mitochondria. The sensitivity of the proline/glutamate antiport to the ionophores valinomycin and nigericin, under conditions in which Dψ and DpH are selectively affected, shows that the exchange is energy dependent. Measurements of both membrane potential and proton movement across the mitochondrial membrane suggest that proline/glutamate antiport is driven by the electrochemical proton gradient via the Dψ dependent proline/glutamate translocator and DpH‐dependent glutamate/OH‐ carrier. Such a carrier provides for re‐uptake of glutamate that has already passed out of the mitochondria in exchange with incoming proline, made possible by the existence of a separate pool of glutamate in the intermembrane space, directly shown by means of HPLC measurements.
Interfacial Air/Water Proton Conduction from Long Distances by Sulfolobus solfataricus Archaeal Bolaform Lipids
Authors: Vilalta Isabelle , Gliozzi Alessandra , Prats Michel 
Published: July 2004

The stability, structural organization, and the ability to transfer protons long distances have been investigated in monolayers formed from archaeal bolaform lipids at the air/water interface. The lipids employed were the fractions GroR2Gro (R represents an acyl group with variable chain length typically consisting of 0–4 cyclopentane rings and 40 isoprenoid residues) and GroR2GroNon‐ol (Non‐ol represents nonitol) extracted from Sulfolobus solfataricus by hydrolysis of the cytoplasmic membrane. GroR2‐GroNon‐ol films exhibit a very peculiar behaviour: the monolayer surface pressure increases with time, regardless of its low or high initial value. This finding is related to the possibility of GroR2GroNon‐ol molecules to assume an upright (a metastable) or a U‐shaped (stable) configuration. In the gaseous state and in the collapsed state of the film, no lateral proton conduction was observed. However, in the pressure range 0 < π < 25 mN/m for GroR2Gro and 0 < π < 30 mN/m for GroR2GroNon‐ol monolayers, a lateral proton conduction at the air/water interface was observed.The structural organization of these bipolar lipids at the air/water interface can be related to the lateral proton conduction; it is possible to conclude that whatever configuration these lipids may adopt, they are able to structure the air/water interface in a hydrogen bond network that supports lateral proton conduction. This process may be ascribed to a percolation phenomenon occurring when the polar lipid head groups form a structured lattice of hydrogen bonds.
Characterization of a Ribosomal Inhibitory Polypeptide of Protein Phosphatase‐1 from Rat Liver
Authors: Beullens Monique , Stalmans Willy , Bollen Mathieu 
Published: July 2004

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase‐1 (PP‐1) and protein phosphatase‐2A (PP‐2A). However, after repeated washing, only PP‐1 remained bound to the ribosomes. The activity of ribosome‐associated PP‐I (PP‐1R) was partially latent and could be increased 2–3‐fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type‐1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP‐1R.We have purified from a ribosomal extract a basic polypeptide (pI ≥ 10.5) of 23 kDa that potently inhibited PP‐1. This ribosomal inhibitor of PP‐1, termed RIPP‐1, was at least 30‐times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP‐2A, PP‐2B and PP‐2C). RIPP‐1 was identified as a non‐competitive inhibitor of PP‐1 with a substrate‐dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type‐1 catalytic subunit, RIPP‐1 also converted the catalytic subunit in a time‐dependent manner (t1/2= 45 min at 25°C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP‐1. We propose that RIPP‐1 accounts, at least in part, for the latency of PP‐1R.
Crystal Structure of Flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in Two Oxidation States
Authors: Romero Antonio , Caldeira Jorge , Legall Jean , Moura Isabel , Moura José J. G. , Romao Maria J. 
Published: July 2004

The crystal structures of the flavodoxin from Desulfovibrio desulfuricans ATCC 27774 have been determined and refined for both oxidized and semi‐reduced forms to final crystallographic R ‐factors of 17.9% (0.8–0.205‐nm resolution) and 19.4% (0.8–0.215‐nm resolution) respectively. Native flavodoxin crystals were grown from ammonium sulfate with cell constants a=b= 9.59 nm, c= 3.37 nm (oxidized crystals) and they belong to space group P3221. Semireduced crystals showed some changes in cell dimensions: a=b= 9.51 nm, c= 3.35 nm. The three‐dimensional structures are similar to other known flavodoxins and deviations are found essentially in the isoalloxazine ring environment. Conformational changes are observed between both redox states and a flip of the Gly61‐Met62 peptide bond occurs upon one‐electron reduction of the FMN group. These changes influence the redox potential of the oxidized/semiquinone couple.Modulation of the redox potentials is known to be related to the association constant of the FMN group to the protein. The flavodoxin from D. desulfuricans now studied has a large span between E2 (oxidized → semiquinone) and E1 (semiquinone → hydroquinone) redox potentials, both these values being substantially more positive within known flavodoxins. A comparison of their FMN environment was made in both oxidation states in order to correlate functional and structural differences.
Mathematical Model for Evaluating the Krebs Cycle Flux with Non‐Constant Glutamate‐Pool Size by 13C‐NMR Spectroscopy
Authors: Tran‐Dinh Son , Beganton Frankie , Nguyen Trong‐Thuc , Bouet Francoise , Herve Martine 
Published: July 2004

A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2‐13C]acetyl‐CoA is not 100% and the total glutamate concentration remains constant or varies during incubation.A comparative study of [1‐13C]glucose metabolism was subsequently carried out using Saccharumyces cerevisiae cells from two different strains (ATCC‐9763 and NCYC‐239) by 13C‐NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2‐oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1‐13C]glucose as a carbon source, the 13C‐NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1‐13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC‐239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl‐CoA, 2‐oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC‐239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.
Kinetics of Phosphorolysis of 3‐(β‐d‐Ribofuranosyl)Adenine and 3‐(β‐d‐Ribofuranosyl)Hypoxanthine, Non‐Conventional Substrates of Purine‐Nucleoside Phosphorylase
Authors: Bzowska Agnieszka , Kulikowska Ewa , Poopeiko Nicolai E. , Shugar David 
Published: July 2004

The properties of two non‐conventional substrates of the calf‐spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3‐(β‐d‐ribofuranosyl)adenine (RibfAde) and 3‐(β‐d‐ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo‐first‐order rate constants (Vmax/Km) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the Corresponding values are 22% and 30%, respectively. The Michaelis constants (Km) for RibfAde were 800 μM (calf PNP) and 150 μM (E. coli PNP). For RibfHyp, the corresponding Km values were 220 μM and 260 μM.Two well‐characterized inhibitors of calf spleen PNP [9‐(2‐fluoro‐3,4‐dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3‐β‐nucleosides occurs at the same active site as for the natural substrate Ino.In particular, the substrate properties of both 3‐β‐nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9‐β‐nucleosides in the conformation anti to the pyrimidine ring.The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.
Microperoxidase/H2O2‐Catalyzed Aromatic Hydroxylation Proceeds by a Cytochrome‐P‐450‐Type Oxygen‐Transfer Reaction Mechanism
Authors: Osman Ahmed M. , Koerts Janneke , Boersma Marelle G. , Boeren Sjef , Veeger Cees , Rietjens Ivonne M. C. M. 
Published: July 2004

The mechanism of aromatic hydroxylation of aniline and phenol derivatives in a H2O2‐driven microperoxidase‐8(MP8)‐catalyzed reaction was investigated. It was shown that the reaction was not inhibited by the addition of scavengers of superoxide anion or hydroxyl radicals, which demonstrates that the reaction mechanism differs from that of the aromatic hydroxylation catalyzed by a horseradish peroxidase/dihydroxyfumarate system.Additional experiments with 18O‐labelled H218O2 demonstrated that the oxygen incoiporated into aniline to give 4‐aminophenol originates from H2O2. Furthermore, it was found that the addition of ascorbic acid efficiently blocks all peroxidase‐type reactions that can be catalyzed by the MP8/H2O2 system, but does not inhibit the aromatic hydroxylation of aniline and phenol derivatives. Together, these observations exclude reaction mechanisms for the aromatic hydroxylation that proceed through peroxidase‐type mechanisms in which the oxygen incorporated into the substrate originates from O2 or H2O. The mechanism instead seems to proceed by an initial attack of the high‐valent iron‐oxo intermediate of MP8 on the π‐electrons of the aromatic ring of the substrate leading to product formation by a cytochrome‐P‐450‐type of σ‐O‐addition or oxygen‐rebound mechanism. This implies that MP8, which has a histidyl and not a cysteinate fifth axial ligand, is able to react by a cytochrome‐P‐450‐like oxygen‐transfer reaction mechanism.
The Structure of the O‐Specific Polysaccharide of Proteus penneri 52
Authors: Sidorczyk Zygmunt , Zych Krystyna , Świerzko Anna , Vinogradov Eugeny V. , Knirel Yuriy A. 
Published: July 2004

An acidic O‐specific polysaccharide isolated from Proteus penneri 52 contains d‐galacturonic acid, 2‐acetamido‐2‐deoxy‐d‐glucose, and 2‐acetamido‐2‐deoxy‐d‐galactose in the ratio 1:2:2. On the basis of sugar analysis and NMR spectroscopy, which included one‐dimensional TOCSY, two‐dimensional COSY, heteronuclear 13C, 1H‐COSY, and rotating‐frame NOE spectroscopy, the following structure of the pentasaccharide repeating unit of the O‐specific polysaccharide was established:
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Cross‐reactivity of the anti‐P‐penneri 52 O‐serum with other strains of P. penneri isolated in Germany, Poland, USA, and Canada is discussed and a new Proteus serogroup is proposed.