bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nitrous oxide reductase from denitrifying Pseudomonas stutzeri
Authors: ZUMFT Walter G. , VIEBROCK‐SAMBALE Adelheid , BRAUN Cornelia 
Published: March 2005

Nitrous oxide (N2O) respiration by the denitrifying bacterium Pseudomonas stutzeri requires the synthesis of the multi‐copper enzyme N2O reductase. nosZ, the structural gene for this enzyme, is part of a DNA region of approximately 8 kbp that carries several essential genes. Insertional mutagenesis of the region downstream of nosZ generates apoenzyme‐synthesizing strains, which argues for the existence of functions for copper acquisition or copper processing, or both, for N2O reductase. The relevant DNA region of approximately 3.2 kbp was sequenced and found to consist of three genes, nosDFY, presumably within a single transcriptional unit. Cellular concentration, copper content, and location of the N2O reductase protein were studied with mutants which were affected in the three genes.Interactions of the deduced gene products among each other and with the cytoplasmic membrane appear to be analogous to those of the components of osmotic‐shock‐sensitive bacterial transport systems. The 33.8‐kDa product of the nosF gene belongs to the family of nucleotide‐binding proteins [C. Higgins et al. (1986) Nature 323, 448–450]. Its amino acid sequence shows two canonical nucleotide‐binding motifs, and the positional identity of amino acids to members of this family is around 30%. The 29.4‐kDa product of the nos Y gene is a hydrophobic protein with six predicted transmembrane helices and an export signal. The 48.2‐kDa product of the nosD gene is a periplasmic component; it carries an export signal and is a hydrophilic protein. N2O reductase itself is a periplasmic enzyme. Our results provide evidence for an auxiliary system of Cu processing and suggest its involvement in the periplasmic biosynthesis of the Cu centers of N2O reductase.
Expression of argininosuccinate lyase mRNA in foetal hepatocytes
Authors: HUSSON Annie , RENOUF Sylvie , FAIRAND Alain , BUQUET Catherine , BENAMAR Malika , VAILLANT René 
Published: March 2005

Argininosuccinate lyase (ASL), the fourth enzyme of the urea cycle, belongs to a group of liver enzymes appearing in the late foetal period in the rat. Several hormones, including glucocorticosteroids and insulin have been implicated in the control of the development of this enzyme activity. In this study, the cloned cDNA was used to measure the relative abundance of ASL mRNA in the livers of rats at various stages of perinatal development and in cultured foetal hepatocytes during hormonal manipulations.The ASL mRNA was first detectable on day 15.5 of gestation and increased in amount concomitantly with the rise in the enzyme activity, suggesting that the appearance of enzyme activity reflects the turning on of specific gene transcription. When foetal hepatocytes were exposed to dexamethasone, an increase in ASL mRNA was detected, which was completely abolished by addition of actinomycin D, suggesting a transcriptional effect of the steroid. In contrast, administration of cortisol to foetuses in utero had no effect on the mRNA level, suggesting that the steroid action is inhibited in the intra‐uterine environment. Insulin might be the inhibiting factor since it completely repressed the dexamethasone‐induced accumulation of ASL mRNA in foetal hepatocytes. These data were confirmed in vivo by experiments using streptozotocin, which produces insulin‐depleted foetuses and causes the accumulation of ASL mRNA. This regulation of ASL mRNA by glucocorticoids and insulin could account for the modulation of the enzyme activity observed in vivo and in vitro.
Purification and characterization of DNA ligase II from Drosophila melanogaster
Authors: TAKAHASHI Miho , TOMIZAWA Kayoko 
Published: March 2005

Drosophila melanogaster contains DNA ligases I and II. The activity of DNA ligase I is especially high during early embryonic periods, but decreases rapidly afterwards. Although the activity of DNA ligase II is low, it persists throughout all developmental stages. The specific activity of DNA ligase II is high in embryos, but the total activity per body mass was highest in pupae. To characterize the properties of DNA ligase II further and to clarify its differences from DNA ligase I, DNA ligase II was prepared from pupae of D. melanogaster. The enzyme was purified about 3200‐fold by ammonium sulfate fractionation (40–70% saturation), phosphocellulose (P11) and Ultrogel column chromatography. Some of the properties have been reported previously. The isoelectric point of DNA ligase II was 6.4 while those of DNA ligase I were 4.9 and 5.8. The optimum pH of DNA ligase II was 7.8–8.1 but 8.0–8.5 for DNA ligase I.The molecular masses of DNA ligase II adducts with AMP were determined as 90 and 70 kDa. These adducts were degraded to 42 and 14.4 kDa by trypsin digestion. For preparation of monoclonal antibodies, a mouse was immunized with the purified enzyme. Two clones, 10‐6 and 3‐3 IgM, were obtained and purified from mouse ascites. These antibodies showed both binding and neutralizing activities toward DNA ligase II from D. melanogaster, but did not react with DNA ligase I from the same origin.These results showed clearly that DNA ligases I and II have different properties and suggest they have different roles during the developmental stages of D. melanogaster.
Biotransformation of intracellular minerals by zinc ions in vivo and in vitro
Authors: TAYLOR Marina G. , GREAVES G. Neville , SIMKISS Kenneth 
Published: March 2005

Inorganic granules of the general type CaMgP2O7 are found within cells of a wide range of species. The granules are amorphous to X‐rays and occur in membrane‐bound compartments. They accumulate a variety of cations from the body fluids of these animals.The interaction of zinc ions with pyrophosphate granules from cells of the snail Helix aspersa have been studied in vivo and in vitro by X‐ray absorption spectroscopy and X‐ray diffraction. The in vivo deposits incorporate zinc and remain amorphous although the pyrophosphate is hydrolysed to orthophosphate. In the in vitro reaction the mineral becomes crystalline but there is little change in the form of the anion. The results clearly indicate a role for the cellular system in maintaining the amorphous state and in the hydrolysis of pyrophosphate to orthophosphate. As such they provide a model system for some of the changes observed in precursors of bone mineral and in pathological changes in the calcification of cartilage.
Ontogeny and perinatal modulation of gene expression in rat brown adipose tissue
Authors: GIRALT Marta , MARTIN Immaculada , IGLESIAS Roser , VIÑAS Octavi , VILLARROYA Francesc , MAMPEL Teresa 
Published: March 2005

We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit II of cytochrome‐c oxidase (COII) and lipoprotein lipase in brown adipose tissue during the fetal and postnatal periods. Moreover, we have determined whether these parameters can be modulated by ambient temperature in the early hours after birth, and at which point in development this sensitivity first appears.UCP gene expression in the fetal and neonatal period has particular features when compared with overall mitochondriogenesis (COII mRNA expression) or with the expression of lipoprotein lipase mRNA. There is a specific induction of UCP gene expression between days 18 and 19 of pregnancy followed by a specific increase of UCP gene expression in utero and a further increase after birth. The acquisition of the physiological apparatus capable of the response to UCP and lipoprotein lipase gene expression to the environmental temperature is not achieved until the last day of fetal development. This result suggests that mechanisms of β‐adrenergic modulation of gene expression in brown fat are already established at birth.From an experiment on iopanoic acid treatment of pregnant mothers, it was concluded that iodothyronine 5′‐deiodinase activity is not necessary for the expression of the mRNAs for UCP, COII and lipoprotein lipase in the fetus whereas it is necessary for the acquisition of temperature sensitivity to these parameters at birth.
Structural and functional aspects of calcium homeostasis in eukaryotic cells
Authors: PIETROBON Daniela , Di VIRGILIO Francesco , POZZAN Tullio 
Published: March 2005

The maintenance of a low cytosolic free‐Ca2+ concentration, ([Ca2+]i) is a common feature of all eukaryotic cells. For this purpose a variety of mechanisms have developed during evolution to ensure the buffering of Ca2+ in the cytoplasm, its extrusion from the cell and/or its accumulation within organelles. Opening of plasma membrane channels or release of Ca2+ from intracellular pools leads to elevation of [Ca2+]i; as a result, Ca2+ binds to cytosolic proteins which translate the changes in [Ca2+]i into activation of a number of key cellular functions.The purpose of this review is to provide a comprehensive description of the structural and functional characteristics of the various components of [Ca2+]i homeostasis in eukaryotes.
Binding and activation of plasminogen at the surface of Staphylococcus aureus
Authors: KUUSELA Pentti , SAKSELA Olli 
Published: March 2005

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I‐Glu‐plasminogen. The binding was inhibited by both unlabeled Glu‐plasminogen and Glu‐plasmin. The Lys form of plasminogen, which lacks the 8‐kDa amino‐terminal activation peptide, was approximately 100‐fold more effective than the Glu form in competing with the binding of 125I‐labeled Glu‐plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. l‐Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine‐binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high‐ and low‐affinity binding sites. Kd and the number of high‐affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I‐Glu‐plasminogen bound to staphylococcal surface was converted to plasmin by tissue‐type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low‐molecular‐mass plasmin inhibitors such as aprotinin, the bound Glu‐plasmin was further converted to Lys‐plasmin. The surface‐bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S‐2251.The plasminogen conversion shown by the present experiments not only leads to the surface‐bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface‐bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface‐bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.
Structural characterization of sialylated tetrasaccharides and pentasaccharides with blood group H and Lex activity isolated from bovine submaxillary mucin
Authors: SAVAGE Angela V. , DONOHUE John J. , KOELEMAN Carolien A. M. , van den EIJNDEN Dirk H. 
Published: March 2005

In this study we have investigated the structures of a sialylated tetrasaccharide and two sialylated pentasaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high‐performance liquid chromatography. The tetrasaccharide contained NeuGc, while one of the pentasaccharides contained NeuAc and the other contained NeuGc. All three oligosaccharides contained the core type‐3 structure (GlcNAcβ1→3GalNAcol). The structures, determined by a combination of one‐ and two‐dimensional 1H‐NMR spectroscopy at 270 MHz and methylation analysis involving gas‐liquid chromatography/mass spectrometry, were as follows:

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The oligosaccharides occurred in the approximate molar ratios, 1.0:0.6:0.3. This is the first report of these oligosaccharides in bovine submaxillary mucin. 1H‐NMR data for structures A1/2c and A1/2e, which are novel structures, are presented for the first time. Oligosaccharide A1/2e contains the blood‐group‐H type‐2 antigenic determinant while oligosaccharide A1/2d contains the Lewisx determinant.
Ascorbyl free radical as a reliable indicator of free‐radical‐mediated myocardial ischemic and post‐ischemic injury
Authors: PIETRI Sylvia , CULCASI Marcel , STELLA Lucien , COZZONE Patrick J. 
Published: March 2005

The real‐time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous‐flow ESR using high‐speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous‐flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH= 0.188 mT and g= 2.0054).

The results showed that ascorbyl free radical release occurred mainly during sequences of low‐flow ischemia (90 min) coupled or not with 30 min of zero‐flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl‐free‐radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen‐derived‐free‐radical‐induced cellular damage.
The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia.
Perfusion of superoxide dismutase during low‐flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free‐radical‐induced myocardial injury.
The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.
A ΔφH clamp method for analysis of steady‐state kinetics of photophosphylation
Authors: STROTMANN Heinrich , THELEN Renate , MÜLLER Wolfgang , BAUM Wolfgang 
Published: March 2005

An instrumental device is described which allows steady‐state kinetic measurements of photophosphorylation at adesired proton gradient which can be maintained throughout the course of the experiment (‘Δφ clamp’). This is achieved by electronic regulation of light intensity using the calibrated 9‐aminoacridine fluorescence signal as sensor of the gradient. The instrument is suitable for determination of kinetic parameters of the proton‐translocating ATPase in isolated envelope‐free chloroplasts under defined conditions.At clamped Δφ, phosphorylation as a function of substrate concentration shows Michaelis‐Menten kinetics. The true Michaelis constantsand the dissociation constants for phosphate and ADP are reported. The Michaelis constants are not affected by the magnitude of the proton gradient in the investigated range. The significance of these results is discussed.
What metabolite levels may be evolutionarily reached in the glycolytic pathway?
Authors: PETTERSSON Gösta 
Published: March 2005

The thermodynamic and kinetic properties of the glycolytic pathway have been analyzed in order to characterize the ultimate limits for the metabolite concentrations that may be evolutionarily reached in response to a selective pressure in the direction of increased glycolytic reaction flux. The results indicate that the chemical potential for conversion of glucose into pyruvate at presently observed levels of these two metabolites (and of NADH, NAD+, ATP, ADP and inorganic phosphate) is high enough to allow ultimately for an accumulation of fructose 1,6‐bisphosphate at concentrations which may be considered as indefinitely high from a practical point of view. This means that non‐kinetic factors such as metabolite solubility put a definite limit to the glycolytic reaction flux that may be evolutionarily reached.With the reasonable assumption that evolution eventually may raise the concentration of fructose 1,6‐bisphosphate minimally to the same level as that presently attained by glucose, analytical evidence is presented to show that catalytic improvement of enzymes in the glycolytic pathway ultimately may result in glycerone phosphate levels that are at least two orders of magnitude higher than those presently observed. This argues strongly against the idea that triose‐phosphate isomerase represents a perfectly evolved catalyst in the sense that it should have reached the end of its evolutionary development.
Photoaffinity labelling of the purine – cytosine permease of Saccharomyces cerevisiae
Authors: CHIRIO Maria‐Chantal , BRÈTHES Daniel , NAPIAS Christian , GRANDIER‐VAZEILLE Xavier , RAKOTOMANANA Félicien , CHEVALLIER Jean 
Published: March 2005

8‐Azidoadenine was used as a photoaffinity reagent to characterize the purine–cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells.In the strain with the amplified purine–cytosine permease gene the maximum cytosine uptake rate was increased 4–5‐fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild‐type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine–cytosine permease.
Metabolism of cysteinyl leukotrienes in monkey and man
Authors: HUBER Michael , MÜLLER Juliane , LEIER Inka , JEDLITSCHKY Gabriele , BALL Howard A. , MOORE Kevin P. , TAYLOR Graham W. , WILLIAMS Roger , KEPPLER Dietrich 
Published: March 2005

The proinflammatory cysteinyl leukotrienes are inactivated in primates by (a) intravascular degradation, (b) hepatic and renal uptake from the blood circulation, (c) intracellular metabolism of leukotriene E4 (LTE4), and (d) biliary and renal excretion of LTC4 degradation products. We have analyzed cysteinyl leukotriene metabolites excreted into bile and urine of the monkey Macaca fascicularis and of man. In both species, hepatobiliary leukotriene elimination predominated over renal excretion. In a representative healthy human subject at least 25% of the administered radioactivity were recovered from bile and 20% from urine within 24 h. In monkey and man intravenous administration of 14,15‐3H2‐labeled LTC4 resulted in the biliary and urinary excretion of labeled LTE4, ω‐hydroxy‐LTE4, ω‐carboxy‐LTE4, ω‐carboxy‐tetranor‐ dihydro‐LTE4, and ω‐carboxy‐tetranor‐dihydro‐LTE4. Small amounts of N‐acetyl‐LTE4 were detected in human urine only. Oxidative metabolism of LTE4 proceeded more rapidly in the monkey resulting in the formation of higher relative amounts of ω‐oxidized leukotrienes in this species as compared to man. [3H]H2O amounted to less than 2% of the administered dose in monkey and human bile and urine samples. Incubation of isolated human hepatocytes with [3H2]LTC4, [3H2]LTE4, and [3H2]LTD4 showed that only [3H2]LTE4 underwent intracellular oxidative metabolism resulting in the formation of ω‐ and β‐oxidation products. N‐Acetylated LTE4 derivatives were not detected as products formed by human hepatocytes.By a combination of reversed‐phase high‐performance liquid chromatography and radioimmunoassay, endogenous LTE4 and N‐acetyl‐LTE4 were detected in human urine in concentrations of 220 ± 40 and 24 ± 3 pM, corresponding to 12 ± 1 and 1.5 ± 0.2 nmol/mol creatinine, respectively (mean ± SEM; n= 10). Endogenous LTD4 and LTE4 were detected in human bile (n= 3) in concentrations between 0.2–0.9 nM. Our results demonstrate that LTD4 and LTE4 are major LTC4 metabolites in human bile and/or urine and may serve as index metabolites for the measurement of endogenously generated cysteinyl leukotrienes. Moreover, ω‐oxidation and subsequent β‐oxidation from the ω‐end contribute to the metabolic degradation of LTE4 not only in monkey but also in man.
Eukaryotic DNA replication
Authors: THÖMMES Pia , HÜBSCHER Ulrich 
Published: March 2005

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. Model systems for replication, such as the simian virus 40 DNA, lead the way.Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double‐helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase δ and the lagging strand discontinuously in small pieces by DNA polymerase α. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3′→ 5′‐exonuclease, mostly part of the DNA polymerase δ polypeptide, can perform proof‐reading by excising incorrectly base‐paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5′→ 3′‐exonuclease, removing the RNA primers, DNA polymerase α or β, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher‐order structures like chromatin.