bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Stabilization of Topoisomerase‐II–DNA Cleavable Complexes by Doxorubicin and Etoposide in Doxorubicin‐Resistant Rat Glioblastoma Cells
Authors: Montaudon Danièle , Pourquier Philippe , Denois Françoise , De Tinguy‐Moreaud Éliane , Lagarde Philippe , Robert Jacques 
Published: July 2004

Using the technique of alkaline filter elution, we have evaluated the DNA damage induced by doxorubicin and etoposide in a rat glioblastoma cell line, C6, and its doxorubicin‐selected resistant variant, C6 0.5. DNA damage paralleled ding‐induced cytotoxicity, but it appeared that the same DNA damage generated much less cytotoxicity in resistant cells than in sensitive ones, resistant cells being able to tolerate more DNA damage than sensitive cells.We have then quantified the doxorubicin‐ and etoposide‐induced complexes between topoisomerase II (topoll) DNA with the technique of SDS/KCl precipitation. Etoposide produced a concentration‐dependent increase in topoII‐DNA complexes, which was higher in resistant cells at equitoxicity, just as was DNA damage. In contrast, doxorubicin‐induced topoII‐DNA complexes, which were much less abundant than those induced by etoposide, were not differently produced in sensitive and resistant cells. This indicates that the DNA damage occurring in resistant cells at high doxorubicin concentrations might originate from source other than topoII‐DNA complex formation.When verapamil was added during drug exposure, it restored doxorubicin intracellular accumulation to the level reached in sensitive cells, partially reversed both doxorubicin and etoposide resistance, increased the formation of etoposide‐induced topoII‐DNA complexes, but not those induced by doxorubicin. Immunoblot analysis of topoll as well as the measure of its catalytic activity in nuclear extracts revealed a quantitative defect of this enzyme in the resistant line. When inhibiting this activity by doxorubicin and etoposide, we observed that the concentrations of etoposide required for a given inhibition of kinetoplast DNA decatenation are much higher that those of doxorubicin. The topoII extracted from both cell lines is, therefore, much more sensitive to doxorubicin than to etoposide, but no difference in drug sensitivity was evident between sensitive and resistant cells, indicating that no qualitative alteration in topoll catalytic activity was likely to occur.
Comparative In‐Vivo and In‐Vitro99Mo‐Time‐Differential‐Perturbed‐Angular‐Correlation Studies on the Nitrogenase Mofe Protein and on Other Mo Species of Different N2‐Fixing Bacteria
Authors: Müller Achim , Suer Waltraud , Pohlmann Carsten , Schneider Klaus , Thies Wolf‐Gerolf , Appel Helmut 
Published: July 2004

Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99MoO2‐4. The distribution of 99Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion‐exchange (DEAE‐Sephacel) chromatography. Cells of K. pneumoniae took up MoO2‐4 only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo‐containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo‐storage protein. In the presence of 20 mM NH4+, conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo‐storage protein exclusively. This protein proved to be unstable towards DEAE‐Sephacel, apparently releasing all the molybdenum in form of MoO2‐4 during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not‐yet‐identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase.The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99Mo(β−)99Tc using time differential perturbed angular correlation spectroscopy. The quadrupole coupling constant (VQ) determined for the Mo center in MoFe proteins was consistently in the range 66–81 MHz. The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement. For the Mo‐storage protein of A. vinelandii, a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase‐repressed cells as well as with gel‐filtered cell‐free extracts. Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro.
Multiheme Cytochromes from the Sulfur‐Reducing Bacterium Desulfuromonas Acetoxidans
Authors: Pereira Inês A. C. , Pacheco Isabel , Liu Ming‐Y. , Legall Jean , Xavier António V. , Teixeira Miguel 
Published: July 2004

Two new multiheme cytochromes were isolated from the anaerobic sulfur reducing bacterium Desulfuromonas acetoxidans. They have monomeric molecular masses of 50 and 65 kDa and contain six and eight hemes, respectively. Visible and EPR spectroscopies, in the as‐isolated (oxidised) cytochromes, show the presence of only low‐spin hemes in the 50‐kDa cytochrome, and of high‐spin and low‐spin hemes in the 65‐kDa cytochrome. The EPR spectra of the native 65‐kDa cytochrome indicate multiple heme–heme interactions, including integer‐spin systems as judged by parallel‐mode EPR. The 50‐kDa cytochrome has a complex redox pattern, as shown by EPR redox titrations, and contains one heme with unusual characteristics. Both cytochromes cover an extremely wide range of reduction potentials, which go from +100 mV to ‐375 mV for the 50‐kDa cytochrome, and +185 mV to ‐235 mV for the 65‐kDa cytochrome.The two cytochromes were tested for hydroxylamine oxidoreductase activity and polysulfide reductase activity, but neither displayed any activity. In contrast, it was found for the first time that the previously characterised cytochrome c551.5, from the same bacterium is very active in the reduction of polysulfide, which suggests that it acts as a terminal reductase in D. acetoxidans.
Concentration of, and SDS Removal from Proteins Isolated from Multiple two‐Dimensional Electrophoresis Gels
Authors: Hatt Paola Dainese , Quadroni Manfredo , Staudenmann Werner , James Peter 
Published: July 2004

We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two‐dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non‐ionic detergent (octyl β‐glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis.The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two‐dimensional gels to 90% for fluorescence and Coomassie‐blue‐stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver‐stained or fluorescent‐stained proteins since the amount of N‐terminal blockage appears to increase as the amount of protein isolated from the two‐dimensional gel decreases.
Steady‐State and Picosecond‐Time‐Resolved Fluorenscence Studies on the Recombinant Heme Domain of Bacillus megaterium Cytochrome P‐450
Authors: Khan Kishore Kumar , Mazumdar Shyamalava , Modi Sandeep , Sutcliffe Mike , Roberts Gordon C. K. , Mitra Samaresh 
Published: July 2004

The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P‐450BM3 have been investigated by steady‐state and picosecond‐time‐resolved fluorescence spectroscopy. The steady‐state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme. substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme. This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate. The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent‐accessible area for the free enzyme agrees with the values obtained by the quenching experiments.The time‐resolved fluorescence studies carried out by means of the time‐correlated single‐photon‐counting technique show that the fluorescence‐decay curve is best fitted to a three‐exponential model (0.2, 1.0 and 5.4 ns). Lifetime distributions, as recovered by the maximum‐entropy method, agree with the discrete exponential model. The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate‐binding domain. The decay‐associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component (τ1) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and τ2 originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent. X‐ray crystallographic data and solvent‐acessible‐area calculations have been used to identify these residues.
Structural Studies on the Short‐Chain Lipopolysaccharide of Vibrio Cholerae O139 Bengal
Authors: Knirel Yuriy A. , Widmalm Göran , Senchenkova Sof'ya N. , Jansson Per‐Erik , Weintraub Andrej 
Published: July 2004

A Vibrio cholerae O139 strain, MO10‐T4, lacking capsular polysaccharide, produces a short‐chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high‐performance anion‐exchange chromatography, one‐ and two‐dimensional 1H‐, 13C‐, and 31P‐NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS:
image

where PEtn stands for 2‐aminoethyl phosphate, Fru for fructose, Hep for l‐glycero‐d‐manno‐heptose, and Kdo for 3‐deoxy‐d‐manno‐octulosonic acid; unless otherwise stated, the monosaccharide residues are d and present in the pyranose form. An O‐acetyl group is present on a secondary position, tentatively O4 of the α‐linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown.An O139‐negative mutant strain, Bengal‐2R, derived from V. cholerae O139, has also been investigated and shown to produce an O‐antigen‐lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10‐T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2‐aminoethyl phosphate, the O‐acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10‐T4 LPS, a short O‐side chain is attached at position 3 of the 7‐substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.‐R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43–58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59–65.]
Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
Authors: Fritsche Erich , Humm Andreas , Huber Robert 
Published: July 2004

L‐Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active‐site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom‐binant human L‐arginine : glycine amidinotransferase were prepared by site‐directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28‐nm, 0.29‐nm and 0.236‐nm resolution, respectively. The mutation of active‐site residues which are involved in substrate‐binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000‐fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild‐type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition‐state stabilization, and not due to major conformational rearrangements of the protein.These results show that a His‐Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain‐like cysteine proteinases.
Preferential Esterification of Endogenously Formed 5‐Hydroxyeicosatetraenoic Acid to Phospholipids in Activated Polymorphonuclear Leukocytes
Authors: Arai Masayoshi , Imai Hirotaka , Metori Atsuko , Nakagawa Yasuhito 
Published: July 2004

The esterification of endogenously formed 5‐hydroxyeicosatetraenoic acid (5‐HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5‐HETE was observed after a 5‐min stimulation of PMNL with A23187. The amount of free 5‐HETE then declined rapidly, while that of 5‐HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time‐dependent manner. Stimulation by A23187 yielded approximately 100 ng/107 cells esterified 5‐HETE in 60 min, which corresponded to the decrease in the amount of free 5‐HETE from 5 min to 60 min and indicated that free 5‐HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids.The esterification profile of exogenous 5‐HETE was different from that of endogenous 5‐HETE. 5‐[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed that endogenous 5‐HETE was esterified equally to phospholipids and triacylglycerol.Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5‐HETE. However, less 5‐HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12‐HETE, produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation.Exogenous 5‐HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12‐HETE into macrophages was observed. The rapid uptake of exogenous 5‐HETE was strongly inhibited by the suppression of acylation of 5‐HETE by triacsin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5‐HETE.
Dissection of the Domain Architecture of the α2macroglobulin‐Receptor‐Associated Protein
Authors: Ellgaard Lars , Holtet Thor Las , Nielsen Peter Reinholt , Etzerodt Michael , Gliemann Jørgen , Thøgersen Hans Christian 
Published: July 2004

The α2macroglobulin‐receptor‐associated protein (RAP) binds to the α2macroglobulin receptor/low‐density lipoprotein receptor‐related protein (α2MR/LRP), a multi‐functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known α2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands.In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39‐kDa protein important for regulating ligand binding to the low‐density lipoprotein receptor‐related protein, Biochemistry 34, 3404–3415], to three structural domains, 1, 2 and 3, comprising residues 18–112, 113–218 and 219–323 of RAP, respectively. Structural analysis by 1H‐NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of α‐helices, whereas domain 3 as a separate domain appears only to be marginally stable.Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I‐RAP and 125I‐α2M* (methylamine‐activated α2M) for binding to α2MR*LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C‐terminal regions of domains 1 and 3.
Structural Studies of the O‐Antigenic Polysaccharide from Escherichia Coli O167
Authors: Linnerborg Malin , Wollin Ralfh , Widmalm Göran 
Published: July 2004

The structure of the O‐antigenic polysaccharide from Escherichia coli O167: H5 has been investigated. Sugar and methylation analyses, fast‐atom‐bombardment mass spectrometry and 1H‐ and 13C‐NMR spectroscopy were the main methods used. The structure of the repeating unit of the polysaccharide was found to be:
image

Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation. Furthermore, base treatment of the polysaccharide led to a degraded polymeric material. For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3‐O‐methyl galactose derivative.
Anaerobic Metabolism of Aromatic Compounds
Authors: Heider Johann , Fuchs Georg 
Published: July 2004

Aromatic compounds comprise a wide variety of low‐molecular‐mass natural compounds (amino acids, quinones, flavonoids, etc.) and biopolymers (lignin, melanin). They are almost exclusively degraded by microorganisms. Aerobic aromatic metabolism is characterised by the extensive use of molecular oxygen. Monoxygenases and dioxygenases are essential for the hydroxylation and cleavage of aromatic ring structures. Accordingly, the characteristic central intermediates of the aerobic pathways (e.g. catechol) are readily attacked oxidatively. Anaerobic aromatic catabolism requires, of necessity, a quite different strategy. The basic features of this metabolism have emerged from studies on bacteria that degrade soluble aromatic substrates to CO2, in the complete absence of molecular oxygen.Essential to anaerobic aromatic metabolism is the replacement of all the oxygen‐dependent steps by an alternative set of novel reactions and the formation of different central intermediates (e.g. benzoyl‐CoA) for breaking the aromaticity and cleaving the ring; notably, in anaerobic pathways, the aromatic ring is reduced rather than oxidised. The two‐electron reduction of benzoyl‐CoA to a cyclic diene requires the cleavage of two molecules of ATP to ADP and Pi and is catalysed by benzoyl‐CoA reductase. After nitrogenase, this is the second enzyme known which overcomes the high activation energy required for reduction of a chemically stable bond by coupling electron transfer to the hydrolysis of ATP. The alicyclic product cyclohex‐1,5‐diene‐1‐carboxyl‐CoA is oxidised to acetyl‐CoA via a modified β‐oxidation pathway; the ring structure is opened hydrolytically. Some phenolic compounds are anaerobically transformed to resorcinol (1,3‐dihydroxybenzene) or phloroglucinol (1,3,5‐trihydroxybenzene). These intermediates are also first reduced and then as alicyclic products oxidised to acetyl‐CoA.This review gives an outline of the anaerobic pathways which allow bacteria to utilize aromatics even in the absence of oxygen. We focus on previously unknown reactions and on the enzymes characteristic for such novel metabolism.
Quantitation of Metabolic Compartmentation in Hyperammonemic Brain by Natural Abundance 13C‐NMR Detection of 13C‐15N Coupling Patterns and Isotopic Shifts
Authors: Lapidot Aviva , Gopher Asher 
Published: July 2004

In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2‐oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains.The 15N enrichments of brain metabolite a‐amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N‐labeled compounds of the 13C‐15N spin coupling patterns of natural abundance 13C‐NMR spectra. The 13C‐NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, l‐methionine DL‐sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5‐15N]glutamine and [2‐15N]glutamine concentrations reached 5.2 μmol/100 mg protein and 3.6 μmol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2‐oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2‐15N]glutamate, which is about 30% of that of [2‐15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by l‐methionine dl‐Sulfoximine, a flux of 15NH4+: via the residual activity of GS was accompanied by an apparent increase of [2‐15N]glutamate and [15N]alanine concentrations (2.9 μmol/100 mg protein and 1.8 μmol/100 mg protein, respectively).These findings and those obtained from 13C‐13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2‐oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.