bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of O‐linked glycosylation patterns between species
Authors: Mourad Rabih , Morelle Willy , Neveu André , Strecker Gérard
Published: March 2003

Amphibia egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O‐linked carbohydrate chains of the jelly coats surrounding the eggs of Rana ridibunda were released by alkaline borohydride treatment. Fractionation of the mixture of O‐linked oligosaccharide‐alditols was achieved by a combination of chromatographic techniques including gel‐permeation chromatography, ion‐exchange chromatography and high‐performance liquid chromatography using an amino‐bonded silica column. The primary structures of these O‐glycans were determined by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy and matrix‐assisted laser‐desorption‐ionization‐time‐of flight mass spectrometry. 25 oligosaccharide structures, possessing a core consisting of Gal(β1–3)GalNAc‐ol with or without branching through a GlcNAc residue linked (β1–6) to the GalNAc residue (core type 2 or core type 1, respectively) are described. The most representative antennae are: HSO3(6)[Fuc(α1‐3)]GlcNAc; Gal(β1‐2)Gal; Gal(β1‐2)Gal(α1‐3)[Fuc(α1‐2)]Gal; GlcA(β1‐3)‐Gal(β1‐3)[Fuc(α1‐2)]Gal; GalNAc(α1‐4)Gal(β1‐4)Gal; Gal(β1‐3)GalNAc(α1‐4)Gal(β1‐4)Gal and GlcA(β1‐3)Gal(β1‐3)GalNAc. These results confirm the species‐specific O‐glycosylation of Amphibia oviducal mucins. The significance of this observation should be linked to a symbiotic role of carbohydrates involved in host–parasite interactions.
Glucose‐6‐phosphate dehydrogenase‐6‐phosphogluconolactonase
Authors: Clarke Julia L. , Scopes Deborah A. , Sodeinde Olugbemiro , Mason Philip J.
Published: March 2003

Plasmodium falciparum glucose 6‐phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N‐terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6‐phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine‐rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full‐length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.
Defined sequence segments of the small heat shock proteins HSP25 and αB‐crystallin inhibit actin polymerization
Authors: Wieske Martin , Benndorf Rainer , Behlke Joachim , Dölling Rudolf , Grelle Gerlinde , Bielka Heinz , Lutsch Gudrun
Published: March 2003

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N‐terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine αB‐crystallin showed the same behaviour. The results suggest that both HSP25 and αB‐crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.
Human phosphatidylinositol 4‐kinase isoform PI4K92
Authors: Suer Silke , Sickmann Albert , Meyer Helmut E. , Herberg Friedrich W. , Heilmeyer Ludwig M. G.
Published: March 2003

Human phosphatidylinositol 4‐kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 ≈ 300 nm) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI‐MS and LC‐MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C‐terminal catalytic domain was designated phosphorylation domain.
Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein
Authors: Isenberg Bärbel , Thole Hubert , Tümmler Burkhard , Demmer Annette
Published: March 2003

P‐glycoprotein is an ATP‐dependent drug‐efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P‐glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P‐glycoprotein‐containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity‐labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I‐labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino‐acid regions 248–312 [transmembrane segment (TM)4 to TM5], 758–800 (beyond TM7 to beyond TM8) and 1160–1218 (after the Walker A motif of the second nucleotide‐binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.
Kinetic and crystallographic analysis of complexes formed between elastase and peptides from β‐casein
Authors: Wright Penny A. , Wilmouth Rupert C. , Clifton Ian J. , Schofield Christopher J.
Published: March 2003

Human β‐casomorphin‐7 (NH2‐Tyr‐Pro‐Phe‐Val‐Glu‐Pro‐Ile‐CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl‐enzyme complex stable enough for X‐ray crystallographic analysis at pH 5. To investigate the importance of the N‐terminal residues of the β‐casomorphin‐7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated β‐casomorphin‐7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri‐, tetra‐and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N‐acetyl‐Val‐Glu‐Pro‐Ile‐CO2H and PPE at pH 5 (to 1.67 Å resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/β‐casomorphin‐7 structure supportive of its assignment as the ‘hydrolytic water’ in the deacylation step of serine protease catalysis.
Sp1 involvement in the 4β‐phorbol 12‐myristate 13‐acetate (TPA)‐mediated increase in resistance to methotrexate in Chinese hamster ovary cells
Authors: Noé Véronique , Alemany Cristina , Nicolás Marta , Ciudad Carlos J.
Published: March 2003

4β‐Phorbol 12‐myristate 13‐acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA‐mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA‐treated cells reduced the binding of Sp1. Stable transfectants of PKCα showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy‐five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCα increased DHFR activity. Over‐expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.
Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase
Authors: Schnorpfeil Michaela , Janausch Ingo G. , Biel Simone , Kröger Achim , Unden Gottfried
Published: March 2003

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2‐(heptyl)‐4‐hydroxyquinoline‐N‐oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N′‐dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Δψ = −132 mV) which was comparable to that generated by glucose oxidation with O2 (Δψ = −120 mV). The Δψ generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half‐loop.
Paralogous gene analysis reveals a highly enantioselective 1,2‐O‐isopropylideneglycerol caprylate esterase of Bacillus subtilis
Authors: Dröge Melloney J. , Bos Rein , Quax Wim J.
Published: March 2003

Carboxylesterase NP of Bacillus subtilis Thai I‐8, characterized in 1992 as a very enantioselective (S)‐naproxen esterase, was found to show no enantiopreference towards (S)‐1,2‐O‐isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)‐IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)‐IPG.
Sequence‐dependent binding of bis‐amidine carbazole dications to DNA
Authors: Tanious Farial A. , Wilson W. David , Patrick Donald A. , Tidwell Richard R. , Colson Pierre , Houssier Claude , Tardy Christelle , Bailly Christian
Published: March 2003

The conventional wisdom argues that DNA intercalators possess a condensed polyaromatic ring whereas DNA minor groove binders generally contain unfused aromatic heterocycles, frequently separated by amide bonds. Recently, this view has been challenged with the discovery of powerful intercalating agents formed by unfused aromatic molecules and groove binders containing a polyaromatic nucleus. Bis‐amidinocarbazoles belong to this later category of drugs having a planar chromophore and capable of reading the genetic information acessible within the minor groove of AT‐rich sequences [Tanious, F.A., Ding, D., Patrick, D.A., Bailly, C., Tidwell, R.R. & Wilson, W.D. (2000) Biochemistry39, 12091–12101]. But in addition to the tight binding to AT sites, we show here that bis‐amidinocarbazoles can also interact with GC sites. The extent and mode of binding of 2,7 and 3,6 substituted amidinocarbazoles to AT and GC sequences were investigated by complementary biochemical and biophysical methods. Absorption, fluorescence, melting temperature and surface plasmon resonance (SPR) measurements indicate that the position of the two amidine groups on the carbazole ring influences significantly the drug–DNA interaction. SPR and DNase I footprinting data confirm the AT‐preference of the compounds and provide useful information on their additional interaction with GC sequences. The 3,6‐carbazole binds approximately twice as strongly to the GC‐containing hairpin oligomer than the 2,7‐regioisomer. The high tendency of the 3,6 compound to intercalate into different types of DNA containing G·C base pairs is shown by electric linear dichroism. This work completes our understanding of the sequence‐dependent DNA binding properties of carbazole dications.
Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide‐lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85
Authors: Vinogradov Evgeny , Egbosimba Emmanuel E. , Perry Malcolm B. , Lam Joseph S. , Forsberg Cecil W.
Published: March 2003

The polysaccharides from the outer membrane of the Gram‐negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size‐exclusion chromatography in the presence of deoxycholate detergent into a lower‐molecular‐mass fraction designated ‘glycolipid’ and a high‐molecular‐mass ‘capsular polysaccharide’ fraction. Both fractions lacked typical lipopolysaccharide components including 2‐keto‐3‐deoxyoctulosonic acid and 3‐hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures:

where HEAEP is N‐(2‐hydroxyethyl)‐2‐aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except l‐rhamnose had a d‐configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide‐less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.
Molecular cloning of bovine zona pellucida glycoproteins ZPA and ZPB and analysis for sperm‐binding component of the zona
Authors: Yonezawa Naoto , Fukui Norio , Kuno Masako , Shinoda Mitsutaka , Goko Satoshi , Mitsui Sachiko , Nakano Minoru
Published: March 2003

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N‐acetyllactosamine at the nonreducing portion of sugar chains with endo‐β‐galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta1201, 7–14]. The N‐terminal amino‐acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino‐acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino‐acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala‐Asp‐Asp/Glu. We purified bovine zona components and examined their sperm‐binding activity with an in vitro competition assay and sperm‐bead‐binding assay. As a result, ZPB showed the strongest sperm‐binding activity among the components. ZPC also showed sperm‐binding activity and the activity per molecule was about one‐sixth that of ZPB according to the result of the sperm‐bead‐binding assay. We could not determine if ZPA has significant sperm‐binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.
Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies
Authors: Poulas Konstantinos , Eliopoulos Elias , Vatzaki Efstratia , Navaza Jorge , Kontou Maria , Oikonomakos Nikos , Acharya K. Ravi , Tzartos Socrates J.
Published: March 2003

The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 Å resolution by X‐ray crystallographic methods. The mouse anti‐lysozyme Fab D1.3 was used as a search model in molecular replacement with the amore software. The complementarity determining regions (CDR)‐L2, CDR‐H1 and CDR‐H2 belong to canonical groups. Loops CDR‐L3, CDR‐H2 and CDR‐H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen‐binding are examined. The antigen‐binding site was found to be a long crescent‐shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis.
Cloning and sequencing of a molluscan endo‐β‐1,4‐glucanase gene from the blue mussel, Mytilus edulis
Authors: Xu Bingze , Janson Jan‐Christer , Sellos Daniel
Published: March 2003

Using polymerase chain reaction, cloning and sequencing techniques, a complementary DNA encoding a low molecular mass cellulase (endo‐1,4‐β‐d‐glucanase, EC 3.2.1.4) has been identified in the digestive gland of the marine mussel, Mytilus edulis. It contains a 5′ untranslated region, a 633‐nucleotide ORF encoding a 211 amino‐acid protein, including a 17 amino‐acid signal peptide and a complete 3′ untranslated region. At the C‐terminal end of the purified mature protein, a 13 amino‐acid peptide is lacking in comparison to the protein sequence deduced from the ORF. This peptide is probably removed as a consequence of post‐translational amidation of the C‐terminal glutamine. The endoglucanase genes have been isolated and sequenced from both Swedish and French mussels. The coding parts of these two sequences are identical. Both genes contain two introns, the positions of which are conserved. However the length of the introns are different due to base substitutions, insertions or deletions showing the existence of interspecies length polymorphism. The percentage of similarity for the introns of the two gene sequences is 96.9%. This is the first time a molluscan cellulase is characterized at DNA level. Amino acid sequence‐based classification has revealed that the enzyme belongs to the glycosyl hydrolase family 45 [B. Henrissat (Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France), personal communication]. There is no cellulose binding domain associated with the sequence.
Modulation of the lipid binding properties of the N‐terminal domain of human apolipoprotein E3
Authors: Weers Paul M. M. , Narayanaswami Vasanthy , Ryan Robert O.
Published: March 2003

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low‐density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130–150 in the independently folded, 22‐kDa N‐terminal (NT) domain. This elongated globular four‐helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro‐PCho) vesicle transformation assays, 100 µg apoE3 NT induced only 15% reduction in vesicle (250 µg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro‐PCho vesicles was concentration‐dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT‐mediated vesicle transformation rates were enhanced > 10‐fold compared with Myr2Gro‐PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT‐mediated Myr2Gro‐PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8‐anilino‐1‐naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.
In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae
Authors: Martins Ana Margarida , Mendes Pedro , Cordeiro Carlos , Freire Ana Ponces
Published: March 2003

The kinetics of glyoxalase I [(R)‐S‐lactoylglutathione methylglyoxal‐lyase; EC 4.4.1.5] and glyoxalase II (S‐2‐hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program hyperfit. Glyoxalase I exhibited saturation kinetics on 0.05–2.5 mm hemithioacetal concentration range, with kinetic parameters Km 0.53 ± 0.07 mm and V (3.18 ± 0.16) × 10−2 mm·min−1. Glyoxalase II also showed saturation kinetics in the Sd‐lactoylglutathione concentration range of 0.15–3 mm and Km 0.32 ± 0.13 mm and V (1.03 ± 0.10) × 10−3 mm·min−1 were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program gepasi. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis–Menten model. The kinetic parameters obtained, Km 0.62 ± 0.18 mm and V (2.86 ± 0.01) × 10−2 mm·min−1, were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis–Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.
A family of novel, acidic N‐glycans in Bowes melanoma tissue plasminogen activator have L2/HNK‐1‐bearing antennae, many with sulfation of the fucosylated chitobiose core
Authors: Zamze Susanne , Wing David R. , Wormald Mark R. , Hunter Ann P. , Dwek Raymond A. , Harvey David J.
Published: March 2003

A family of about 20 novel acidic bi‐ and tri‐antennary N‐glycans, amounting to almost half those expressed on Bowes melanoma tissue‐plasminogen activator (t‐PA) were found to possess Galβ1→4GlcNAcβ1→, sulfated and sialylated GalNAcβ1→4GlcNAcβ1→ or sulfated GlcAβ1→ 3Galβ1→4GlcNAcβ1→ antennae, of which those containing sulfated GlcA, depicting the L2/HNK‐1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK‐1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6‐branched antenna and an α2→6 sialylated GalNAc on the other. The N‐glycosylation sequon containing Asn448, which is known to express all of the sulfate‐carrying N‐glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core‐sulfate group of the N‐glycan is proposed to reduce mobility of the carbohydrate in the region of the t‐PA active site. Because of the ‘brain‐type’ nature of the N‐glycans described in this neuro‐ectodermal cell line, the possibility of neural t‐PA interacting with the L2/HNK‐1‐recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.
Probing the conformational state of a truncated staphylococcal nuclease R using time of flight mass spectrometry with limited proteolysis
Authors: Yang Feng , Cheng Yuan , Peng Jiarou , Zhou Junmei , Jing Guozhong
Published: March 2003

The conformational state of C‐terminally truncated staphylococcal nuclease R (SNR135), with and without bound ligands, has been studied by performing limited proteolysis with a specific endoproteinase Glu‐C followed by electrophoresis and mass spectrometry. Comparison of the accessibility of the cleavage sites shows that the C‐terminal truncation of 14 amino‐acid residues causes significant unfolding of the C‐terminal part of α helix 1 and the center of α helix 2, but there is little effect on other regions of the nuclease, in particular the N‐terminal subdomain, which includes the active site of the nuclease. The truncation also makes the overall conformation of the nuclease more loose and flexible. Binding of ligands makes helices 1 and 2 more resistant to protease Glu‐C attack and converts the partially unfolded state to a native‐like state, although the conformational stability of the SNR135 complex is still much lower than that of the full‐length enzyme. The results suggest that the amino‐acid residues around the active site in the truncated nuclease are arranged in a similar topology to those in the full‐length nuclease. The study shows that there is a clear‐cut correlation between protease susceptibility and conformational stability of the protein, and the initial proteolytic events are the most critical for evaluating the conformational features of the protein. This study demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.
Novel hopanoids from Frankia spp. and related soil bacteria
Authors: Rosa‐Putra Surya , Nalin Renaud , Domenach Anne‐Marie , Rohmer Michel
Published: March 2003

Three series of hopanoids, differing by their configurations at C‐17 and C‐21, have been identified in several Frankia spp. and other related soil bacteria. The widespread bacterial hopanoids of the 17β(H),21β(H) series were accompanied by their isomers of the 17β(H),21α(H) (moretane) and 17α(H), 21β(H) series. The latter series has not previously been found in living organisms and is considered to be a result of the abiotic isomerization of the thermodynamically less stable 17β(H),21β(H) hopanoids. This simultaneous presence of three isomeric hopanoid series highlights intriguing problems in the biogenesis of the bacteriohopane skeleton and partly questions the significance of hopanic biomarkers in sediments.
Characterization of the formyltransferase from Methylobacterium extorquens AM1
Authors: Pomper Barbara K. , Vorholt Julia A.
Published: March 2003

Methylobacterium extorquens AM1 possesses a formaldehyde‐oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate‐reducing archaea. Here we describe the purification and characterization of formylmethanofuran–tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5‐formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 µm; apparent Km for H4MPT = 30 µm). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of ≈ 40% with Ftr from methanogenic and sulfate‐reducing archaea. The 32‐kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active‐site cofactors molybdenum, molybdopterin and a [4Fe−4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.
Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type‐1
Authors: Wind Troels , Jensen Mads A. , Andreasen Peter A.
Published: March 2003

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase‐serpin complex implies insertion of the reactive centre loop of the serpin into the large central β‐sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including α‐helix F. In order to elucidate this mechanism, we have used phage‐display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against α‐helix F and its flanking region in the serpin plasminogen activator inhibitor‐1 (PAI‐1). One of these is known to inhibit the reaction between PAI‐1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI‐1. When combined with the effects these antibodies have on PAI‐1 activity, our epitope mapping points to the mobility of amino‐acid residues in α‐helix F and the loop connecting α‐helix F and β‐strand 3A as being important for the inhibitory function of PAI‐1. Although all antibodies reduced the affinity of PAI‐1 for vitronectin, the potentiating effect of vitronectin on antibody‐induced PAI‐1 neutralization is based on formation of a ternary complex between antibody, PAI‐1 and vitronectin, in which PAI‐1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI‐1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI‐1 in cancer and cardiovascular diseases.
High affinity binding between laminin and laminin binding protein of Leishmania is stimulated by zinc and may involve laminin zinc‐finger like sequences
Authors: Bandyopadhyay Keya , Karmakar Sudipan , Ghosh Abhijit , Das Pijush K.
Published: March 2003

In the course of trying to understand the pathogenesis of leishmaniasis in relation to extracellular matrix (ECM) elements, laminin, a major ECM protein, has been found to bind saturably and with high affinity to a 67‐kDa cell surface protein of Leishmania donovani. This interaction involves a single class of binding sites, which are ionic in nature, conformation‐dependent and possibly involves sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys‐rich zinc finger‐like sequences on laminin. Inhibition studies with monoclonals against polypeptide chains and specific peptides with adhesive properties revealed that the binding site was localized in one of the nested zinc finger consensus sequences of B1 chain containing the specific pentapeptide sequence, YIGSR. Furthermore, incubation of L. donovani promastigotes with C(YIGSR)3‐NH2 peptide amide or antibody directed against the 67‐kDa laminin‐binding protein (LBP) induced tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa. These studies suggest a role for LBP in the interaction of parasites with ECM elements, which may mediate one or more downstream signalling events necessary for establishment of infection.
The Fe‐only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus
Authors: Siemann Stefan , Schneider Klaus , Dröttboom Melanie , Müller Achim
Published: March 2003

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe‐only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox‐titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P‐cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell‐shaped redox curve with midpoint potentials (Em) of −195 mV (appearance) and −30 mV (disappearance), (2) an axial signal (g|| = 2.00, g⊥ = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the ‘low‐potential’ rhombic signal and the axial signal have been both attributed to the one‐electron‐oxidized P‐cluster (P1+) present in two conformationally different proteins, the ‘high‐potential’ rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P‐cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: −80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption‐shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite‐reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this ‘turnover signal’ nor any other S = 1/2 signal were detectable. All Rc1Fe‐specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.
The Influence of Repeated Releases on the Homing Behaviour of Sand Martins (Riparia riparia)
Authors: Giunchi Dimitri , Mongini Emanuele , Baldaccini N. Emilio
Published: March 2003

Data on the effect of repeated releases on the homing behaviour of sand martins released four times from the same site (beyond 50 km from the nesting colony) are presented. The results, obtained by two series of releases, show a progressive improvement of both the initial orientation and the homing performances. When the birds released four times from the same site were displaced in a new site, located roughly at the same distance, but in the opposite direction to the former, they do not change directional preferences with respect to the last experimental release; the homing speeds were also not different to those recorded for birds without any release experience. While the learning of orienting cues can not be excluded, the data point out that the habituation to the handling stress and a form of directional training seem to play a major role in determining the observed behaviour. The familiarity with a site does not seem to improve the homing behaviour of sand martins when released from a different one.
Female Reproductive Cycle and Sexual Conflict over Precopulatory Mate‐guarding in Thermosphaeroma (Crustacea, Isopoda)
Authors: Jormalainen Veijo , Shuster Stephen M.
Published: March 2003

In species with time‐limited opportunities for insemination, precopulatory mate‐guarding is expected to coevolve with the duration of female reproductive cycles. Despite this adaptation to female characteristics, it may also be advantageous for males to adjust the duration of guarding with respect to sex ratio because the benefits of guarding are dependent on the availability of females. If female fitness is reduced because of guarding, male guarding behavior leads to intersexual conflict. We studied these aspects of male mate‐guarding behavior in two closely related, thermal‐spring isopods (Thermosphaeroma). First, guarding duration showed species specificity which was related to the duration of reproductive cycle; cycle length for females and duration of guarding by males in T. milleri were twice as long as in T. thermophilum. Second, males in both species adjusted their guarding duration with sex ratio, guarding longer when a competing male was present. Third, in T. thermophilum, ovarian development began immediately after the birth of the previous brood and continued through guarding, sexual molt and post‐molt periods until oviposition, whereas in T. milleri, ovarian development was largely postponed until the post‐molt period. Because guarding during ovary provisioning periods may be costly for females, we tested the existence of intersexual conflict over guarding duration in T. thermophilum. We compared the duration of guarding of control pairs with those of pairs in which either male guarding ability or female ability to resist guarding was reduced experimentally. Guarding durations for manipulated and control males were equal, but manipulated females were guarded longer, suggesting that conflict exists and that females can effectively shorten guarding duration by their behavior. Moreover, we suggest that selection in the context of intersexual conflict may play an important role in the evolution of delayed oviposition and sperm‐storage organs in mate‐guarding crustaceans.
Chemical Recognition of Familiar vs. Unfamiliar Conspecifics by Juvenile Iguanid Lizards, Ctenosaura similis
Authors: Hanley Kathryn A. , Elliott Meredith L. , Stamps Judy A.
Published: March 2003

The ability of juvenile black iguanas, Ctenosaura similis, to discriminate between the chemical cue of an unfamiliar and a familiar conspecific was tested. Lizards directed significantly more tongue extrusions toward the chemical cue of a size‐matched conspecific with whom they had never interacted than the chemical cue of a size‐matched conspecific with whom they had interacted for 3 d. We consider the possibility that the social relationship between familiar associates may explain variation in the direction and degree of response to unfamiliar vs. familiar chemical cues.
Assessing the Ideal Free Distribution: Do Guppies Use Aggression as Public Information about Patch Quality?
Authors: Koops Marten A. , Abrahams Mark V.
Published: March 2003

Aggression can be costly to foragers, yet some recent research suggests that foragers should use aggression as a cue to patch quality (the attractive aggression hypothesis). If aggression is predictive of patch quality, then the attractive aggression hypothesis predicts that the distribution of foragers should follow the distribution of aggression. If, instead, aggression is repulsive because it is costly, then the distribution of foragers should diverge from the distribution of aggression. We tested the attractive aggression hypothesis using female guppies, Poecilia reticulata, and found that the distribution of foragers followed the distribution of food, but was unaffected by the distribution of aggression. These data do not support the attractive aggression hypothesis, but instead suggest that the distribution of aggression is a consequence of the distribution of foragers, and that aggression is not used as public information about patch quality.
Comparison of Antipredator Responses of Two Related Water Striders to a Common Predator
Authors: Krupa James J. , Sih Andrew
Published: March 2003

This study compares the antipredator responses of two congeneric water striders that live in different habitats yet share a common predator. Aquarius nebularis and A. remigis are stream‐dwelling species of water striders found in close association in central Kentucky. Aquarius nebularis typically inhabit third‐ and fourth‐order streams that support diverse and complex fish communities; A. remigis inhabit adjacent first‐ and second‐order tributaries that tend to have either no fish or relatively few fish patchily distributed. The ecological conditions for these two species are distinctly different, yet the green sunfish (Lepomis cyanellus) is a surface‐feeding predator and a potential risk to both species. In experimental pools with sunfish, an initial fish strike caused A. remigis to move to the edge of the pool and become inactive. For the rest of the observation period, A. remigis reduced behaviour patterns that attract predators such as skating on open water, mating and male–male aggression. The proportion of A. remigis missing from sunfish pools was 5 times higher than in pools lacking fish, suggesting that 20% of water striders were killed by predation. In contrast, an initial fish strike caused A. nebularis to exhibit a burst of activity as individuals erratically skated over the open water. They then resumed the less active pre‐attack behaviours a short time later. Aquarius nebularis showed none of the antipredator behaviours typical for A. remigis. Furthermore, the proportion of A. nebularis missing from sunfish pools did not differ from that in pools without fish. Although antipredator behaviour has been examined for relatively few species of water striders, this study suggests that the behaviour of the most commonly studied species, A. remigis, may not be indicative for water striders in general., Corresponding author: James J. Krupa, School of Biological Sciences, University of Kentucky, Lexington, KY 40506‐0225, U.S.A. E‐mail:
bio149@pop.uky.edu
bio149@pop.uky.edu
Competition for Milk in the Domestic Rabbit: Survivors Benefit from Littermate Deaths
Authors: Drummond Hugh , Vázquez Elidee , Sánchez‐Colón Salvador , Martinez‐Gómez Margarita , Hudson Robyn
Published: March 2003

We sought evidence for postnatal resource limitation among littermates of the domestic rabbit Oryctolagus cuniculus, and asked whether deaths of indi~vidual pups benefit survivors by increasing their share of milk. Milk ingestion, growth and mortality of 10 chinchilla‐breed captive litters were recorded between birth and age 21 d. That milk limits growth and survival was indicated by larger litters showing lower weight gain and higher mortality, and by a significant positive correlation between milk ingested by individual pups and weight increase. Within litters, pups with higher birth weights grew faster, and weight hierarchies became increasingly stable over the 3 weeks, suggesting that advantages accrued during gestation were progressively consolidated during lactation. After individual pups died, the total daily milk weight obtained by the litter was generally unaffected but per capita milk consumption and growth of surviving pups increased, and increases in per capita consumption were greater in smaller litters. The most successful competitors for milk apparently benefit from the deaths of their littermates by obtaining an increased share of an undiminished daily food supply. This relationship has not previously been demonstrated in any vertebrate.