bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the mechanism of transcellular lipoxin formation in human platelets and granulocytes
Authors: EDENIUS Charlotte , STENKE Leif , LINDGREN Jan Åke 
Published: March 2005

Endogenous arachidonic acid was converted to lipoxins A4, B4 and (6S)‐lipoxin A4, in ionophore‐A23187‐stimulated mixtures of human platelets and granulocytes, while no lipoxins were formed when these cells were incubated separately. However, pure platelet suspensions transformed exogenous leukotriene A4 to lipoxins, including lipoxin A4 and (6S)‐lipoxin A4, but not lipoxin B4. This compound was produced exclusively in the presence of granulocytes.A common unstable tetraene intermediate in lipoxin formation, 15‐hydroxy‐leukotriene A4 [5(6)‐epoxy‐15‐hydroxy‐7,9,13‐trans‐11‐cis‐eicosatetraenoic acid], was indicated by trapping experiments with methanol. Thus, identical profiles of less polar tetraene‐containing derivatives were formed from leukotriene A4 in platelet suspensions, from exogenous 15‐hydroxyeicosatetraenoic acid in granlocyte suspensions and from endogenous substrate in mixed platelet/granulocyte suspensions.Evidence for the involvement of 12‐lipoxygenase in platelet‐dependent lipoxin formation was obtained. Thus, lipoxin synthesis from leukotriene A4 and 12‐hydroxyeicosatetraenoic acid production from arachidonic acid by human platelets was equally inhibited by 15‐hydroxyeicosatetraenoic acid with 50% inhibition obtained at 7.0 μM and 8.2 μM, respectively. In experiments with subcellular preparations from platelets, lipoxin synthesis was observed in both the particulate and soluble fraction and was paralleled by the 12‐lipoxygenase activity. Furthermore, lipoxin formation from leukotriene A4 in platelet sonicates was dose‐dependently inhibited by exogenous arachidonic acid. Finally, 12‐lipoxygenase‐deficient platelets from a patient with chronic myelogenous leukemia were totally unable to produce lipoxins from exogenous or granulocyte‐derived leukotriene A4.It is concluded that the transcellular lipoxin synthesis is dependent on the platelet 12‐lipoxygenase and proceeds via the unstable intermediate, 15‐hydroxy‐leukotriene A4. This tetraene epoxide is transformed to lipoxin B4 by a granulocyte epoxide hydrolase activity or to lipoxin A4 and lipoxins A4/B4 isomers by enzymatic or non‐enzymatic hydrolysis.
Two different aa3‐type cytochromes can be purified from the bacterium Bacillus cereus
Authors: GARCIA‐HORSMAN J. Arturo , BARQUERA Blanca , ESCAMILLA Jose E. 
Published: March 2005

Two aa3‐type cytochromes were purified from membranes of sporulating Bacillus cereus. One of them, an aa3 complex, was found to be composed of two subunits (51 and 31 kDa), two a hemes and three copper atoms, thus being similar to the cytochrome aa3 previously purified from vegetative B. cereus [García‐Horsman, J. A., Barquera, B., González‐Halphen, D. & Escamilla, J. E. (1991) Mol. Microbiol. 5, 197–205]. The second isoform, a caa3 complex, was expressed in sporulating cells only, and was found to be composed of two subunits (51 and 37 kDa). The 37‐kDa subunit (subunit II) is a heme‐c‐containing polypeptide as shown by its peroxidase activity in SDS/PAGE gels and by its spectral features. Both subunits of the caa3 complex immunologically cross‐reacted with antiserum raised against B. cereus cytochrome aa3, suggesting homology between the two enzymes. Also, the heme‐c‐containing subunit of the caa3 complex was reactive with anti‐(bovine cytochrome c) antiserum, but not with anti‐(bovine cytochrome c1) antiserum. In addition to one heme c and two hemes a, the caa3 complex contained three copper atoms.Kinetic comparison of aa3 and caa3 complexes revealed that the latter is slightly more active (k= 150 s−1) and has a lower affinity to yeast cytochrome c (Km= 76 μM) and to oxygen (Km= 2 μM) as compared with cytochrome aa3 (100 s−1, 10 μM, and 5 μM, respectively).
The role of the internal hydrogen bond network in first‐order protein electron transfer between Saccharomyces cerevisiae iso‐1‐cytochrome c and bovine microsomal cytochrome b5
Authors: WHITFORD David , GAO Yuan , PIELAK Gary J. , WILLIAMS Robert J. P. , McLENDON George L. , SHERMAN Fred 
Published: March 2005

An internal water molecule (designated WAT166) is found in iso‐1‐cytochrome c which is part of a redox‐state‐dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site‐directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H‐NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso‐1‐cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild‐type or variant yeast iso‐1‐cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso‐1‐cytochrome c the rate constants measured by saturation transfer are wild‐type (Asn52, Eo= 270 mV, kex= 0.3 s‐1), Asn52 → Ala (Eo= 240 mV, kex= 0.6 s‐1), Asn52 →Ile(Eo=220 mV, kex=1.0 s‐1). The first‐order rates are compared with that of a fourth variant Phe82 → Gly which has been measured previously (Eo=220 mV, kex=0.7 s‐1).An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.
Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C
Authors: SALAMINO Franca , SPARATORE Bianca , DE TULLIO Roberta , MENGOTTI Paola , MELLONI Edon , PONTREMOLI Sandro 
Published: March 2005

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase.Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose‐dependent fashion which results in a linearly correlated decrease in O2‐ production. Taken together, these results provide a direct demonstration that in neutrophils the intracellular level of protein kinase C represents one of the determinants of the rate and extent of O2‐ production.
Purification of penicillin‐binding protein 4 of Escherichia coli as a soluble protein by dye‐affinity chromatography
Authors: MOTTL Harald , KECK Wolfgang 
Published: March 2005

The dacB gene of Escherichia coli, coding for penicillin‐binding protein 4 (PBP4) was cloned under the control of the phage lambda pR promoter and cro gene translation signals. Derepression of the phage lambda promoter for 2 h at 42°C in E. coli led to the maximum over‐production of PBP4 to 3.8° of the total soluble protein. Expression at 42°C but not at 40°C or 37°C led to incomplete processing and aggregation of the preform of PBP4. Cibacron navyblue 2G‐E was selected from a collection of triazine dyes as having a high affinity for PBP4. The immobilised dye was used in a two‐step procedure to isolated 374 mg PBP4 from the soluble fraction of 125 g (wet mass) cells of the over‐producing strain, with a recovery of 63.2° and a final purity of 99° as determined by active‐site titration with radiolabelled penicillin.Saturation of PBP4 with various β‐lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of β‐lactams from the dye matrix.PBP4 behaved as a soluble protein throughout the purification, that was performed in the complete absence of detergents. Furthermore, in flotation experiments on sucrose density gradients and in Triton X‐114 fractionation experiments, it showed the characteristics of a soluble protein. Cibacron navyblue 2G‐E showed class specificity for all E. coli PBP except PBP3 and could be used for the isolation of these PBP from membrane extracts.
Adaptive regulation of Na+‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1
Authors: HELPS Christopher R. , McGIVAN John 
Published: March 2005

1. Na+‐dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein.2. The bovine kidney epithelial cell line NBL‐1 expresses a Na+‐dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low‐phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34±0.18 and 2.95 ± 0.1 nmol · mg‐1 and Km values of 17.1 ± 3.0 and 36.3 ± 7.14 μM for phosphate transport, respectively. Thus there is a significant (P<0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low‐phosphate medium.3. Adaptation of phosphate transport in NBL‐1 cells to low‐phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein.4. Cells were adapted to low‐phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high‐phosphate medium.5. On the basis of these observations, it is proposed that the phosphate transporter of NBL‐1 cells is a 55‐kDa glycoprotein.Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na+‐dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1—3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.
Cell‐free immunity in Cecropia
Authors: BOMAN Hans G. , FAYE Ingrid , GUDMUNDSSON Gudmundur H. , LEE Jong‐Youn , LIDHOLM Dan‐Anders 
Published: March 2005

ContentsIntroduction (Table 1)LysozymeTwo main forms of attacinThe cecropins and their precursor formsChemical synthesis of cecropins and three‐dimensional structure of cecropin ASpectrum and mechanism of action of cecropinsOther factors related to the immune responseGenes for cecropia immune proteinsThe place of synthesis of the immune proteinsDiscussion
The three‐dimensional structure of a DNA hairpin in solution
Authors: BLOMMERS Marcel J. J. , VAN DE VEN Frank J. M. , VAN DER MAREL Gÿs A. , VAN BOOM Jacques H. , HILBERS Cornelis W. 
Published: March 2005

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two‐dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5′ and H5” resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter‐residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation‐matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles β, γ and ɛ were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi‐conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B‐type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained‐molecular‐mechanics as well as free‐molecular‐mechanics calculations are in accordance with those obtained by means of the analysis described above.The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T‐A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B‐type double‐helical stem. The second base of the loop is folded into the minoi groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.
Some properties of site‐specific mutants of human carbonic anhydrase II having active‐site residues characterizing carbonic anhydrase III
Authors: REN Xilin , JONSSON Bengt‐Harald , LINDSKOG Sven 
Published: March 2005

Four amino acid residues, His64, Asn67, Leu198 and Va1207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle‐specific, low‐activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found.A Combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4‐nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 103‐104 times less strongly than isoenzyme II, only [Asn‐67 → Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme‐III‐specific amino acid residues.
Mitochondrial translational‐initiation and elongation factors in Saccharomyces cerevisiae
Authors: VAMBUTAS Andrea , ACKERMAN Sharon H. , TZAGOLOFF Alexander 
Published: March 2005

C155 and E252 are respiratory‐defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild‐type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild‐type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non‐fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2.Disruption of the chromosomal copy of each gene in a wild‐type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFMI gene, coding for the mitochondrial initiation factor, indicates that the amino‐terminal 423 residues of the protein are sufficient to promote peptide‐chain intiation in vivo.
Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin
Authors: Van DAMME Els J. M. , KAKU Hanae , PERINI Fulvio , GOLDSTEIN Irwin J. , PEETERS Ben , YAGI Fumio , DECOCK Benny , PEUMANS Willy J. 
Published: March 2005

Poly(A)‐rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17‐kDa lectin polypeptide upon translation in a wheat‐germ cell‐free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2‐kDa signal peptide.A second post‐translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA.Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal‐sequence and a C‐terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.
Tracking of proton flow during transition from anaerobiosis to steady state
Authors: LUVISETTO Siro , COLA Carmen , SCHMEHL Ibolya , AZZONE Giovanni Felice 
Published: March 2005

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification).2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization.3. The comparison of the bromothymol blue response with the arsenazo III, 2′,7′‐bis(carboxyethyl)‐5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b)phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4.4. Although bromothymol blue always behaves as a membrane‐bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane‐potential‐determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer.5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.
The secondary structure of echistatin from 1H‐NMR, circular‐dichroism and Raman spectroscopy
Authors: SAUDEK Vladimir , ATKINSON R. Andrew , LEPAGE Pierre , PELTON John T. 
Published: March 2005

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine‐rich, RGD‐containing proteins, isolated from the venom of the saw‐scaled viper Echis carinatus.Analysis of circular‐dichroism spectra indicates that, at 20°C, echistatin contains no α‐helix but contains mostly β‐turns and β‐sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40°C and 72°C being irreversible.Raman spectra also indicate considerable β‐turn and β‐sheet (20%) structure and an absence of α‐helical structure. Three of the four disulphide bridges are shown to be in an all‐gauche conformation, while the fourth adopts a trans‐gauche‐gauche conformation.The 1H‐NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27°C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either α‐helical or β‐sheet conformation. A number of turns could be characterised. An irregular β‐hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross‐links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.
A calorimetric study of the Ni(II)‐5′AMP system
Authors: HERRERO Luis A. , CALAFAT Antònia M. , TERRÓN Angel 
Published: March 2005

The thermodynamic parameters of the interaction between Ni(II) (M) and 5′‐AMP in aqueous solution were determined calorimetrically (I= 0.1 tetramethylammonium bromide, 25°C) at physiological pH. The experimental conditions were carefully selected to avoid the polynuclear complex formation. From the Ni(5′AMP)‐complex‐formation constant (logK1= 2.55), the Ni(5′AMP)2−2‐complex‐formation constant and the Ni(5′AMP) (ML) and Ni(5′AMP)2−2 (ML2) complex‐formation enthalpies were determined (logK2= 2.34, ΔH1=–10.0 kJ/mol and ΔH2= 21.6 kJ/mol).These results confirm the formation of the ML2 complex in solution and agree very well with the hypothesis of the ‘stacking’ between the purine rings, promoted for metal ions.