bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequencing, Expression and Properties of Triosephosphate Isomerase from Entamoeba Histolytica
Authors: Landa Abraham , Rojo‐Domínguez Arturo , Jiménez Lucia , Fernández‐velasco D. Alejandro 
Published: July 2004

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved poly‐peptide sequences, derived from TPIs of other organisms, were used to amplify a 450‐bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43–52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild‐type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N‐terminal tail of six histidine residues E. histolytica TPI‐His6); both recombinant proteins were purified.Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.
Identification of A Putative Histidine Base and of a Non‐Protein Nitrogen Ligand in the Active Site of Fe‐Hydrogenases by One‐Dimensional and Two‐Dimensional Electron Spin‐Echo Envelope‐Modulation Spectroscopy
Authors: Van Dam Pieter J. , Reijerse Eduard J. , Hagen Wilfred R. 
Published: July 2004

The active H‐cluster of the Fe‐hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one‐ and two‐dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen‐containing ligand was found. The unusual quadrupole interaction parameters (D. vulgaris: quadrupole coupling constant, K= 1.20 MHz, asymmetry parameter η= 0.32, M. elsdenii: K= 1.23 MHz, η= 0.25) indicate a non‐protein type of nitrogen and are consistent with cyanide as ligand to the H‐cluster.The additional interactions measured on the EPR signal of the inactivated H‐cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske‐type iron‐sulfur clusters. Since a His residue near the putative H‐cluster binding motif of Cys residues, His371, is the only conserved His in Fe‐hydrogenases, it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The inactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H‐cluster.
Structural Elucidation of two Polysaccharides Present in the Lipopolysaccharide of a Clinical Isolate of Burkholderia Cepacia
Authors: Cérantola Stéphane , Montrozier Henri 
Published: July 2004

Based on the sugar composition, methylation analyses and Smith degradation, supported by NMR spectroscopic analyses and fast‐atom‐bombardment MS experiments, the lipopolysacharide produced by a clinical isolate of Burkholderia cepacia was shown to contain two distinct polymers, both with linear trisaccharide repeating units; a major, containing D‐rhamnose and D‐galactose residues (2:1) with the structure
image

and a minor repeating unit, constituted by D‐rhamnosyl residues, with the structure
image
The Enzymology of Lysine Catabolism in Rice Seeds — Isolation, Characterization, and Regulatory Properties of a Lysine 2‐Oxoglutarate Reductase/Saccharopine Dehydrogenase Bifunctional Polypeptide
Authors: Gaziola Salete A. , Teixeira Cristiana M. O. , Luoli Juverlande , Sodek Ladaslav , Azevedo Ricardo A. 
Published: July 2004

In plant, the catabolism of lysine has only been studied in some detail in maize. The enzymes lysine α‐oxoglutarate reductase (also known as lysine α‐ketoglutarate reductase; LOR) and saccharopine dehydrogenase (SDH), which convert lysine into saccharopine, and saccharopine into glutamic acid and 2–aminoadipate 6‐semialdehyde, respectively, were isolated from immature rice seeds and partially purified through a three‐step purification procedure involving ammonium sulphate precipitation, and anion‐exchange and gel‐filtration chromatographies, leading to a final yield of 30% for LOR and 24% for SDH. The molecular masses estimated by gel‐filtration chromatography on a Sephacryl S200 column and by native non‐denaturing PAGE using Ferguson plots were 203 kDa for both enzymes by gel‐filtration and 202 kDa for both enzymes by native non‐denaturing PAGE. A second band of LOR and SDH activities on native gels was observed for both enzymes with an estimated molecular mass of 396 kDa, which indicated a multimeric structure. Kinetic studies were consistent with an ordered sequence mechanism for LOR, where 2‐oxoglutarate is the first substrate and saccharopine is the last product.The results observed for the LOR/SDH activity ratios during purification, the copurification in all three steps, the molecular masses, the relative mobilities on native non‐denaturing gels and the pl estimated for LOR and SDH suggest the existence of a bifunctional polypeptide containing LOR and SDH activities.
Activation of Progelatinase B (proMMP‐9) by Active Collagenase‐3 (MMP‐13)
Authors: Knäuper Vera , Smith Bryan , López‐Otin Carlos , Murphy Gillian 
Published: July 2004

Human progelatinase B was activated by collagenase‐3 in a time‐dependent fashion. Activation proceeded through an intermediate form of Mr 86000 to the final active form of Mr 82000. N‐terminal amino acid sequence determination demonstrated that the Glu40–Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87–Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active collagenase‐3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by collagenase‐3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with collagenase‐3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C‐terminal domain interactions between progelatinase B and collagenase‐3, as assessed using wild‐type and C‐terminal deletion mutants of both enzymes.Since elevated levels of both gelatinase B and collagenase‐3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.
Lipoxygenase‐2 Oxygenates Storage Lipids in Embryos of Germinating Barley
Authors: Holtman Wessel L. , Vredenbregt‐Heistek Jolanda C. , Schmitt Nathalie F. , Feussner Ivo 
Published: July 2004

Besides the pre‐existing lipoxygenase (LOX‐1) present in quiescent grains, a new lipoxygenase (LOX‐2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. & Douma, A. C. (1996)Plant Physiol. III, 569–576]. The fact that LOX‐I and LOX‐2 form different products after incubation with linoleic acid, the (9S)‐ and (13S)‐hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991)FEBS Lett. 280, 159–162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. & Douma, A. C. (1992)Biochim. Biophys. Acta 1120, 97–104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX‐I oxygenated at C9, and LOX‐2 at C13. When testing more complex substrates, it was found that both LOX‐1 and LOX‐2 were capable of metabolizing esterified fatty acids. Km values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7‐35‐fold for LOX‐1 versus 2‐8‐old for LOX‐2). Interestingly, LOX‐1 showed significantly higher Km values for esterified fatty acids than did LOX‐2. This was reflected by analyses of the products formed from di‐ and tri‐linoleoylglycerol; LOX‐2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX‐1, with a corresponding larger extent of oxygenation.In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX‐derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX‐2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)‐hydroxy derivatives were formed, indicating the in vivo action of LOX‐2. These data show that LOX‐2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX‐2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)‐13‐hydroxy‐octadecadienoic acid, serves as an endogenous substrate for β‐oxidation and therefore as a carbon source for the growing barley embryo.
The AlkB Monooxygenase of Pseudomonas oleovorans
Authors: Staijen Ivo E. , Hatzimanikatis Vassily , Witholt Bernard 
Published: July 2004

We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk + recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli.The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amounts of AlkB as P. oleovorans (1.5–2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB. The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic‐labeling and immunological techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 μg · mg protein‐−1· h−1, respectively. The half‐lives of AlkB were estimated to be 80, 3 and 15 for P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47), respectively. Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates.The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.
Partial Identification of the Zn2+‐Binding Sites in Factor XII and its Activation Derivatives
Authors: Røjkjær Rasmus , Schousboe Inger 
Published: July 2004

With the purpose of identifying the Zn2+–binding sites in factor XII, the effect of chemical modification of His, Glu and Asp residues, amino acids known to participate in the catalytic coordination binding of Zn2+ in a number of Zn2+–binding proteins, was analysed. The number of modifiable His residues in factor XII and α–factor XIIa was 16.0±0.7 and 17.3±0.7, respectively. When factor XII/α–factor XIIa was incubated with saturating concentrations of Zn2+ before the diethylpyrocarbonate modification of the His residues, these numbers were reduced to 6.3±0.1 and 8.2±0.5, indicating that ten and nine His residues, respectively, are involved in the binding.Analysis of the Zn2+–binding capacity of factor XII, α–factor XIIa and β–factor XIIa showed that while factor XII contains four Zn2+–binding sites, α–factor XIIa had only three and β–factor XIIa had none. Modification of the His residues resulted in a complete loss of Zn2+‐binding while Asp/Glu modification resulted in loss of two and one Zn2+‐binding sites in factor XII and α‐factor XIIa, respectively. This suggests that two of the four sites in factor XII contain His residues, exclusively, while the two others are comprised of two His residues and one Asp/Glu residue. One of the latter is lost when factor XII is activated to α‐factor XIIa. Two of the sites are suggested to be located at positions His40‐His44 and His78–His82. The location of the remaining two sites are reduced to four possible positions.
Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by ΔH+
Authors: Cappellini Paolo , Turina Paola , Fregni Valeria , Melandri B. Andrea 
Published: July 2004

Sulfite stimulates the rate of ATP hydrolysis by the ATP synthase in chromatophores of Rhodobacter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in uncoupled chromatophores. The activation consists in an increase of about 12–15‐fold of the Vmax for the ATP hydrolysis reaction, while the Km for MgATP is unaffected at 0.16±0.03 mM. The dependence of Vmax on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12 mM.Sulfite affects the ability of the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP‐induced protonic flows demonstrate that at sulfite concentrations of greater than 10 mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by light or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentrations of at least 80 mM.Sulfite interferes with the mechanism of activation by ΔH+. Low concentrations of this anion (≤ 2 mM) prevent the activation by ΔH+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a ΔH+ across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite.These experimental results can be simulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K1S= 0.26 and K2S= 37 mM, and for phosphate K1P= 0.06 and K2S= 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is consistent with the molecular structures of the two ligands and of the enzyme.
NADH Oxidase Activity of Human Xanthine Oxidoreductase
Authors: Sanders Stephen A. , Eisenthal Robert , Harrison Roger 
Published: July 2004

Human xanthine oxidase was purified from breast milk. The dehydrogenase form of the enzyme, which predominates in most mammalian tissues, catalyses the oxidation of NADH by oxygen, generating superoxide anion significantly faster than does the oxidase form. The corresponding forms of bovine enzyme behave very similarly. The steady‐state kinetics of NADH oxidation and superoxide production, including inhibition by NAD, by the dehydrogenase forms of both enzymes, are analysed in terms of a model involving two‐stage recycling of oxidised enzyme.Established inhibitors of xanthine oxidoreductases (allopurinol oxypurinol, amflutizole and BOF 4272), which block all other reducing substrates, were ineffective in the case of NADH. Diphenyleneiodo‐nium, on the other hand, was a powerful inhibitor of NADH oxidation.The potential involvement of reactive oxygen species arising from NADH oxidation by xanthine oxidoreductase in ischaemia‐reperfusion injury and other disease states, as well as in normal signal trans‐duction, is discusssed.
Sequence‐Specific Antiproliferative Effects of Antisense and End‐Capping‐Modified Antisense Oligodeoxynucleotides Targeted against the 5′‐Terminus of Basic‐Fibroblast‐Growth‐Factor mRNA in Coronary Smooth Muscle Cells
Authors: Schmidt Annette , Sindermann Jürgen , Peyman Anusch , Uhlmann Eugen , Will David W. , Müller Joachim Georg , Breithardt Günter , Buddecke Eckhart 
Published: July 2004

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate‐modified 15‐residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1–2.0 μM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose‐dependent manner. The effect is sequence specific at low (0.1–0.5 μM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48–72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligo‐nucleotide‐free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15‐residue antisense oligodeoxynucleotide is markedly enhanced by adding 3–4 nonbase‐pairing guanosine residues at the 5′‐ and 3′‐termini of the 15‐residue antisense oligonucleotide.The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF‐specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post‐angioplasty restenosis.
Gloverin, an Antibacterial Protein from the Immune Hemolymph of Hyalophora Pupae
Authors: Axén Andreas , Carlsson Anette , Engström Åke , Bennich Hans 
Published: July 2004

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (PI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins.Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1–3 μM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100°C, 10 min) but is inhibited by Mg2+and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an α‐helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell‐bound LPS.The activity of gloverin is in many respects similar to that of attach, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.‐Y. & Lindholm, D. A. (1991) Eur. J. Biochem. 201, 23–31].
Expression of CYP3A in the Human Liver — Evidence that the Shift between CYP3A7 and CYP3A4 Occurs Immediately After Birth
Authors: Lacroix Dan , Sonnier Michelle , Moncion Arlette , Cheron Gérard , Cresteil Thierry 
Published: July 2004

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P‐450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16α hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6β derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30–40% of the adult activity after one month.CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age.These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.
Structural Studies of the O‐Specific Chains of Hafnia Alvei Strains 744, PCM 1194 and PCM 1210 Lipopolysaccharides
Authors: Petersson Carl , Jachymek Wojciech , Klonowska Agnieszka , Lugowski Czeslaw , Niedziela Tomasz , Kenne Lennart 
Published: July 2004

The structures of the O‐specific chains of the Hafnia alvei strain 744 and PCM strains 1194 and 1210 lipopolysaccharides have been investigated. Methylation analysis, dephosphorylation, NMR spectroscopy, matrix‐assisted laser‐desorption ionisation time‐of‐flight mass spectrometry and fast‐atom‐bombardment mass spectrometry were the principal methods used. It was concluded that the polysaccharides of strains 744 and PCM 1194 are composed of the same pentasaccharide repeating unit having the following structure:
image

and that the polysaccharide of strain PCM 1210 is composed of a pentasaccharide repeating unit having the following structure:
image