bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Partial Characterisation of a Reversible Artificial Mediator Accepting NADH Oxidoreductase from Clostridium thermoaceticum
Authors: Bayer Manfred , Walter Katrin , Simon Helmut 
Published: July 2004

An NAD(H)‐dependent artificial mediator accepting pyridine nucleotide oxidoreductase present in Clostridium thermoaceticum has been purified 50‐fold by three chromatographic steps to apparent electro‐phoretical homogeneity with a yield of 25%. By PAGE and gel filtration the molecular mass of the native enzyme was estimated to be 200 kDa and 210 kDa, respectively. By SDS/gel electrophoresis, a single band was found at 17000 Da, suggesting a homododecamer. Reducing carbamoyl methylviologen or hexa‐cyanoferrate(III) with NADH, the enzyme was most active at pH 10 and the specific activities were 100 μmol min‐1 mg‐1 protein and 800 μmol min‐1 mg‐1 protein, respectively. The Km values for hexacya‐noferrate(III), carbamoylmethylviologen and NADH at pH 8.5 were determined to be 0.40, 0.55 and 1.1 mM, respectively. Other electron acceptors for the dehydrogenation of NADH were 2,6‐dichlorophe‐nolindophenol, anthraquinone‐2,6‐disulphonate, ubiquinone 0 and FAD. In the reduction of NAD+ with reduced methyl viologen (MV+), the specific activity was about 225 μmol min‐1 mg‐1 protein at the pH maximum of 5.0. The Km values for reduced methylviologen, NADH and NAD+ were 1.0, 1.1 and 0.25 mM, respectively.The enzyme had 10.6 atoms iron and 12.7 atoms sulphur per dodecamer. A significant content of flavin or molybdopterin cofactor could not be detected.The first 45 amino acids of the oxidoreductase show a surprisingly high degree of identity or similarity with the ribosomal L12 protein of various eubacteria, the acyl carrier proteins of microorganisms, but also with bovine heart mitochondria and a 3‐phosphoglycerate dehydrogenase as well as a gyceraldehyde‐3‐phosphate dehydrogenase from bacteria and pea chloroplasts, respectively.
Separate Compartments for the Production of Sterols, Carotenoids and Gibberellins in Gibberella fujikuroi
Authors: Domenech Carlos Eduardo , Giordano Walter , Ávalos Javier , Cerdá‐olmedo Enrique 
Published: July 2004

Substrate flows in the sterol, carotenoid and gibberellin pathways of Gibberella fujikuroi were examined by isotope‐dilution experiments. The wild type and two carotenoid mutants of this fungus were grown in minimal medium with abundant glucose, limiting ammonium nitrate and a radioactively labelled precursor (either acetate, mevalonate or leucine). The precursors did not affect growth or terpenoid production, with two exceptions; leucine allowed additional growth, as expected from the nitrogen limitation in the medium, and mevalonate inhibited the accumulation of gibberellins, but only if added before the onset of gibberellin production.The relative contributions of glucose, mevalonate, leucine and acetate as terpenoid precursors, calculated from the specific radioactivities of ergosterol, neurosporaxanthin and phytoene, were different for different products and different precursors. We conclude that the biosyntheses of sterols, gibberellins and carotenoids in Gibberella are physically separated in different subcellular compartments with independent substrate pools. The same results were obtained with the three strains, except for carotenoid production, indicating that this pathway is regulated independently from other terpenoid pathways.
The Binding of Nucleotides to Domain I Proteins of the Proton‐Translocating Transhydrogenases from Rhodospirillum rubrum and Escherichia coli as Measured by Equilibrium Dialysis
Authors: Bizouarn Tania , Diggle Christine , Jackson J. Baz 
Published: July 2004

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)‐binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherichia coli were overexpressed and purified. Nucleotide binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd= 32 μM and 120 μM, respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers, and at pH 7.5 in sodium phosphate buffer. NAD+ bound with lower affinity (Kd= 300 μM). NADPH bound only very weakly (Kd>1 mM).Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M. & Hatefi, Y. (1993) J. Biol. Chem. 268, 17871–17877] found that mitochondria1 transhydrogenase, and a proteolytically derived domain I fragment from that enzyme, bound one NADH per dimer. They suggested that this result implied half‐of‐the‐sites reactivity for the interaction between the nucleotide ligand and the protein. However, our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I proteins using equilibrium dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monomeric units is evident. Reasons for the discrepancies between the work on bacterial and mitochondrial transhydrogenases are discussed.
Molecular‐Dynamics Simulation of Domain Movements in Aspartate Aminotransferase
Authors: Kasper Patrik , Sterk Michael , Christen Philipp , Gehring Heinz 
Published: July 2004

Mitochondrial aspartate aminotransferase is a homodimeric protein with 2×402 amino acid residues. The enzyme in solution undergoes ligand‐induced and syncatalytic conformational changes which appear to correspond to shifts in the equilibrium between the crystallographically defined open and closed conformation. In the closed conformation, the small domain of each subunit has rotated as a rigid body by 13° and 14° towards the large coenzyme‐binding domain and has closed the active‐site pocket.Molecular dynamics simulations at 300 K of 120‐ps duration were started from the crystal structures of the unliganded pyridoxal form (open form) of the dimeric enzyme and the 2‐methylaspartate‐liganded closed form in which the 2‐methyl group had been removed. Both structures contained the crystal water molecules and were placed in a 5‐Å shell of water. The rms fluctuations of the individual Cα atoms during the simulations agreed well with the corresponding B factors of the crystal structures. Superposition of the initial structures and the average structures of the last 20 ps showed in both simulations extensive Cα deviations in the case of the whole subunit but much smaller changes in the individual large and small domains, indicating a movement of the two domains relative to each other. In the simulation of the open form, superposition of the large domains made evident a displacement of the small domain towards its position in the closed crystal structure, which can be described by a rotation of the small domain by about 13° around the twofold symmetry (z) axis. A significantly less extensive rearrangement of parts of the small domain, i.e. a rotation of about 5° around the z axis, was observed in the simulation of the substrate‐liganded enzyme (closed form) which, in contrast to the open form, showed only small conformational changes around the active site. In both simulations an additional rotation of the small domain by 9° around the x axis occurred. The actual domain movement is estimated to occur in a time range at least two orders of magnitude larger than the simulation time of 120 ps. Apparently, the surface tension of the unrestrained nonspherical shell of water accelerates the simulated conformational change which, however, quite closely imitates the geometric features of the extensive movement of the small domains (each ≈130 residues).
Mathematical Models for Determining Metabolic Fluxes through the Citric Acid and the Glyoxylate Cycles in Saccharomyces cerevisiae by 13C‐NMR Spectroscopy
Authors: Tran‐Dinh Son , Bouet Françoise , Huynh Quang‐Tru , Herve Martine 
Published: July 2004

We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast. These models are based on the analysis of 13C‐NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched [2‐13C]acetate. The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced.The aerobic metabolism of 100% [2‐13C]acetate in acetate‐grown S. cerevisiae cells was studied as a function of time using 13C‐NMR, 1H‐NMR and biochemical techniques. The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different. By contrast, the glutamate C4 singlet increases then decreases with time. The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t 90 min. A similar observation was made for the C2 resonance singlet and doublet. In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling. These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl‐CoA are negligible in yeast.The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet. The fluxes in the citric acid and the glyoxylate cycles were found to be comparable. The proportion of glutamate in isotopic exchange with the citric acid cycle is about 2.5% min ‐1 in yeast.
Analysis of Global Responses by Protein and Peptide Fingerprinting of Proteins Isolated by Two‐Dimensional Gel Electrophoresis
Authors: Quadroni Manfredo , Staudenmann Werner , Kertesz Michael , James Peter 
Published: July 2004

A set of 8 proteins (SSI, sulfate‐starvation‐induced proteins) was observed by comparative two‐dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two‐dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI 2, 5, 7, 8) to be identified with a high degree of confidence.To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on‐line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5′ region of the Escherichia coli genome. N‐terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product. SSI 4 was not found in the databases by any of these methods.The methods described are of general use for the rapid analysis of complex cell responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N‐terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.
Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNALys3 and Affinity Modification of The Enzyme by tRNALys3 Derivatives
Authors: Nevinsky Georgy A. , Zakharova Ol'ga D. , Fournier Michel , Andreola Marie Line , Litvak Simon , Tarrago‐Litvak Laura 
Published: July 2004

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV‐1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non‐complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70–80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity.In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo‐first‐order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Ki values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.
Molecular Cloning of Limulusα2‐Macroglobulin
Authors: Iwaki Daisuke , Kawabata Shun‐ichiro , Miura Yoshiki , Kato Atsuko , Armstrong Peter B. , Quigley James P. , Nielsen Kåre Lehmann , Dolmer Klavs , Sottrup‐Jensen Lars , Iwanaga Sadaaki 
Published: July 2004

The American horseshoe crab Limulus polyphemus contains α2‐macroglobulin (α2M) in the hemolymph plasma and hemocytes. α2M from Limnulus shows many of the typical characteristics of mammalian α2M, including the presence of an internal thiol‐ester, reactivity with a diversity of endopeptidases, a unique proteinase‐trapping mechanism, and reactivity with the mammalian α2M receptor. Additionally, Limulusα2M has the unique property that it regulates the limulin‐based hemolytic system of the plasma. A cDNA encoding Limulusα2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N‐terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian α2Ms (28–29% identity) and contains common features found in mammalian α2Ms, a bait region, an internal thiol‐ester site, and a receptor‐binding domain. However, the N‐terminal portion (positions 24–105) has no sequence similarity with those of mammalian α2Ms, and it is structurally related to that of the human complement factor C8γ chain, consistent with a role for Limulus α2M in host defense. The component sugar analysis of Limulus α2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian α2M. However, unlike mammalian α2M, no sialic acid was detected in Limulusα2M and it contained approximately 3 mol/mol N‐acetylgalactosamine, suggesting the presence of O‐linked sugar chains, which have not been found in mammalian α2M.Expression of α2M was detected in hemocyies, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against α2M indicated the presence of the α2M in large granules. Trypsin‐treated Limulusα2M, but not the native α2M, displaced methylamine‐treated human 125I‐α2M from the human α2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase‐clearance mechanisms between mammalian and arthropod evolutionary lineages.
Evaluation of (Z)‐9‐tricosene baited targets for control of the housefly (Musca domestica) in outdoor situations
Authors: Hanley M. E. , Dunn D. W. , Abolins S. R. , Goulson D. 
Published: July 2004

Abstract:Houseflies (Musca domestica L.) are a major pest species in a variety of outdoor situations, notably on and around livestock farms and landfill used for the disposal of domestic waste. Currently no effective options are available for control of houseflies outdoors, because many populations exhibit at least some resistance to all available synthetic pesticides. (Z)‐9‐tricosene is the only commercially available pheromone for use in lure‐and‐kill approaches to housefly control, and it is widely used in indoor livestock units in combination with sugar/insecticide bait. Here we examine the potential of this approach for use outdoors, on a landfill site. We investigate the efficacy of toxic targets painted with a sugar/insecticide/(Z)‐9‐tricosene mix. The effects of target size and pheromone concentration were examined in two replicate trials, conducted in June and September 2003. As expected, catch consisted largely of males, was consistently higher on larger traps, and generally increased with (Z)‐9‐tricosene concentration even up to very high levels. However, in repeated trials, and despite mass release of marked flies, catch rates appeared to be insufficient to provide adequate control. We suggest that this is probably because (Z)‐9‐tricosene is primarily a short‐range attractant, and fly populations in outdoor situations are generally distributed over a large area. Catch declined rapidly to zero within 2 weeks, indicating that improved formulation or target design is needed to slow the weathering of active ingredients on the targets. It seems unlikely that (Z)‐9‐tricosene is sufficiently attractive to houseflies to provide an effective and economic lure in outdoor situations.
Antennal and behavioural response of codling moth Cydia pomonella to plant volatiles
Authors: Ansebo L. , Coracini M. D. A. , Bengtsson M. , Liblikas I. , Ramírez M. , Borg‐Karlson A.‐K. , Tasin M. , Witzgall P. 
Published: July 2004

Abstract:Identification of host volatile compounds attractive to codling moth Cydia pomonella, a most important insect of apple, will contribute to the development of safe control techniques. Synthetic apple volatiles in two doses were tested for antennal and behavioural activity in codling moth. Female antennae strongly responded to (Z)3‐hexenol, (Z)3‐hexenyl benzoate, (Z)3‐hexenyl hexanoate, (±)‐linalool and E,E‐α‐farnesene. Two other compounds eliciting a strong antennal response were the pear ester, ethyl (E,Z)‐2,4‐decadienoate, and its corresponding aldehyde, E,E‐2,4‐decadienal, which is a component of the larval defence secretion of the European apple sawfly. Attraction of codling moth to compounds eliciting a strong antennal response was tested in a wind tunnel. Male moths were best attracted to a blend of (E,E)‐α‐farnesene, (E)‐beta‐farnesene and ethyl (E,Z)‐2,4‐decadienoate. The aldehyde E,E‐2,4‐decadienal had an antagonistic effect when added to the above mixture.
Time‐dose‐mortality modelling and virulence indices for six strains of Verticillium lecanii against sweetpotato whitefly, Bemisia tabaci (Gennadius)
Authors: Wang L. , Huang J. , You M. , Liu B. 
Published: July 2004

Abstract:Bioassays of six strains of Verticillium lecanii (Zimmermann) Viégas were conducted with the sweet potato whitefly, Bemisia tabaci. For inoculation, batches of third‐instar whitefly nymphs on sweetpotato seedlings were immersed in conidial suspensions of five dosages at concentrations from 103 to 107 conidia/ml. Each dosage was used to inoculate 150–250 nymphs. The nymphs were maintained at 25°C, under 95% RH, photo phase of 16 : 8 (L : D) and observed daily for mortality. The resulting data were analysed over 8 days by a complementary log‐log (CLL) time‐dose‐mortality model, based on the Hosmer–Lemeshow test, analysing the time‐dose trends for five concentrations of six strains of V. lecanii simultaneously. The parameters from the model were used to estimate the virulence indices (the values of LC50) of six strains against B. tabaci. Based on the time‐dose‐mortality relationships fitted and the virulence indices, the virulence of the six strains of V. lecanii for B. tabai was compared. Results indicated that the strain Vl6063 imported from Canada and the domestic strains V3450 and Vp28 derived from B. tabaci and a scale insect, respectively, were more virulent than the others with LC50 values of 2.57 × 105, 6.03 × 105 and 6.03 × 105, respectively.
How to catch foliage‐dwelling spiders (Araneae) in maize fields and their margins: a comparison of two sampling methods
Authors: Ludy C. , Lang A. 
Published: July 2004

Abstract:The foliage‐dwelling spider fauna was collected in maize fields and on stinging nettles in adjacent margins in Bavaria, South Germany. Two different sampling methods were evaluated: drop cloth sampling and suction sampling. The overall catch was dominated by juvenile spiders, web‐building spiders, and spiders of the families Theridiidae, Linyphiidae, Tetragnathidae and Araneidae (in decreasing order). Field margins harboured more species than maize fields, whereas the total spider abundance was higher in the maize crop. Web‐building spiders such as Theridiidae and Linyphiidae were prominent in maize by individual numbers. Suction sampling with a small suction device proved to be a more efficient and consistent sampling method for foliage‐dwelling spiders than drop cloth sampling. Density and species richness of foliage‐dwelling spiders in maize was shown to be fairly high, implying that spiders of higher strata may play a more important role in biological control than suspected up to now.
Assessment of the abbreviated Duke Social Support Index in a cohort of older Australian women
Authors: Powers Jennifer R , Goodger Brendan , Byles Julie E 
Published: July 2004

Objectives: To assess the acceptability, reliability and validity of the 11‐item Duke Social Support Index (DSSI) in community‐dwelling older Australian women, and to describe its relationship with the women's sociodemographic and health characteristics. Methods: Women aged 70–75 years were randomly selected from the national Medicare database, with over‐sampling of rural and remote areas. The mailed survey included items about social support, Medical Outcomes Study Short Form Health Survey (SF‐36), health service use, recent life events and sociodemographics. Results: All DSSI items were completed by 94% of the 12 939 participants. Internal reliability was reasonable for 10 of the 11 DSSI items and its factors, social interaction (four items) and satisfaction with social support (six items; Cronbach's alpha of 0.8, 0.6, 0.8). The factor structure was consistent for subgroups of women: urban/non‐urban; English speaking/non‐English speaking background; married/widowed. Summed scores were highly correlated with factor scores and showed good construct validity. Higher social support was associated with better physical and mental health, being Australian born, more educated and better able to manage on income. Conclusion: Ten of the 11 DSSI items provided an acceptable, brief and valid measure of social support for use in mailed surveys to community‐dwelling older women.