bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the hatching enzyme from Xenopus laevis
Authors: Fan Ting‐Jun , Katagiri Chiaki
Published: March 2003

Using an anti‐(glutathione S‐transferase–UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel‐filtration and ion‐exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti‐(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40‐kDa form. The 60‐kDa HE had a high hydrolytic and UEVE‐solubilizing activity, and its activities against Boc‐Leu‐Gly‐Arg‐7‐amino‐4‐methylcoumarin (‐NH‐Mec) and UEVE were inhibited by anti‐(GST UVS.2) Ig in a dose‐dependent manner. The 60‐kDa form was easily autodigested into a 40‐kDa form. The 40‐kDa molecule alone had no detectable UEVE‐solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60‐kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60‐kDa molecule into 40‐kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co‐ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc‐Leu‐Gly‐Arg‐NH‐Mec was optimal at pH of 7.4, and with an apparent Km value of 200 µmol·L−1 at 30 °C. The HE is very sensitive to trypsin‐specific inhibitors such as leupeptin, (4‐amidino‐phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N‐α‐tosyl‐l‐lysylchloromethane (Tos‐Lys‐CH2Cl), indicates that it is a trypsin‐type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease‐specific ‘HExHxxGFxHE’ sequence in the deduced HE amino‐acid sequence, indicates that this HE is a Zn2+ metalloprotease.
Solution structure of the lipoyl domain of the chimeric dihydrolipoyl dehydrogenase P64K from Neisseria meningitidis
Authors: Tozawa Kaeko , Broadhurst R. William , Raine Andrew R. C. , Fuller Christopher , Alvarez Anabel , Guillen Gerardo , Padron Gabriel , Perham Richard N.
Published: March 2003

The antigenic P64K protein from the pathogenic bacterium Neisseria meningitidis is found in the outer membrane of the cell, and consists of two parts: an 81‐residue N‐terminal region and a 482‐residue C‐terminal region. The amino‐acid sequence of the N‐terminal region is homologous with the lipoyl domains of the dihydrolipoyl acyltransferase (E2) components, and that of the C‐terminal region with the dihydrolipoyl dehydrogenase (E3) components, of 2‐oxo acid dehydrogenase multienzyme complexes. The two parts are separated by a long linker region, similar to the linker regions in the E2 chains of 2‐oxo acid dehydrogenase complexes, and it is likely this region is conformationally flexible. A subgene encoding the P64K lipoyl domain was created and over‐expressed in Escherichia coli. The product was capable of post‐translational modification by the lipoate protein ligase but not aberrant modification by the biotin protein ligase of E. coli. The solution structure of the apo‐domain was determined by means of heteronuclear NMR spectroscopy and found to be a flattened β barrel composed of two four‐stranded antiparallel β sheets. The lysine residue that becomes lipoylated is in an exposed β turn that, from a {1H}‐15N heteronuclear Overhauser effect experiment, appears to enjoy substantial local motion. This structure of a lipoyl domain derived from a dihydrolipoyl dehydrogenase resembles that of lipoyl domains normally found as part of the dihydrolipoyl acyltransferase component of 2‐oxo acid dehydrogenase complexes and will assist in furthering the understanding of its function in a multienzyme complex and in the membrane‐bound P64K protein itself.
The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5′‐dithiobis(2‐nitrobenzoic acid)
Authors: Kangur Liina , Palumaa Peep
Published: March 2003

The reaction of Cd5Zn2‐metallothionein (MT) with 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 µm concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 ± 2.7 kJ·mol−1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose–response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constants were calculated as follows: ATP, K = 0.31 ± 0.06 mm; ADP, K = 0.26 ± 0.07 mm. Several compounds such as AMP, S‐methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations.
Different effects of tert‐butylhydroperoxide‐induced peroxynitrite‐dependent and ‐independent DNA single‐strand breakage on PC12 cell poly(ADP‐ribose) polymerase activity
Authors: Palomba Letizia , Guidarelli Andrea , Scovassi A. Ivana , Cantoni Orazio
Published: March 2003

The short‐chain lipid hydroperoxide analogue tert‐butylhydroperoxide induces peroxynitrite‐dependent and ‐independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert‐butylhydroperoxide treatment with peroxynitrite‐independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP‐ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP‐ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP‐ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert‐butylhydroperoxide‐induced peroxynitrite‐independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP‐ribose) polymerase.
Engineering the properties of a cold active enzyme through rational redesign of the active site
Authors: Tsigos Iason , Mavromatis Konstantinos , Tzanodaskalaki Maria , Pozidis Charalambos , Kokkinidis Michael , Bouriotis Vassilis
Published: March 2003

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side‐chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild‐type in the temperature range 5–25 °C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5–15 °C and a significantly increased activity at 20–25 °C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135→Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild‐type sequence, which may affect active site flexibility through changes in various conformational constraints.
The effect of changing the hydrophobic S1′ subsite of thermolysin‐like proteases on substrate specificity
Authors: de Kreij Arno , van den Burg Bertus , Veltman Oene R. , Vriend Gert , Venema Gerard , Eijsink Vincent G. H.
Published: March 2003

The hydrophobic S1′ subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin‐like protease (TLP) produced by Bacillus stearothermophilus (TLP‐ste), the hydrophobic S1′ subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild‐type S1′ pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1′ position. Rather unexpectedly, the Leu202→Phe and Leu202→Tyr mutations, which were expected to decrease the size of the S1′ subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1′ position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1′ subsite. Thermolysin and TLP‐ste variants with identical S1′ subsites were highly similar in terms of their preference for Phe vs. Leu in the P1′ position.
Molecular characterization of a phosphatidylcholine‐hydrolyzing phospholipase C
Authors: Preuss Ilka , Kaiser Iris , Gehring Ulrich
Published: March 2003

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine‐hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl‐phosphatidylcholine and 1‐palmitoyl‐2‐arachidonoyl‐phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of ≈ 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine‐specific PLCs, caused half‐inhibition of the P. fluorescens enzyme at ≈ 420 µm, while 50‐fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.
Cloning and expression of a membrane‐bound CMP‐N‐acetylneuraminic acid hydroxylase from the starfish Asterias rubens
Authors: Martensen Ilka , Schauer Roland , Shaw Lee
Published: March 2003

The sialic acid N‐glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP‐Neu5Ac hydroxylase. The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning. Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found. The unusual characteristics of this hydroxylase raise questions about its evolution. As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme. Despite many similarities with its mammalian counterpart, CMP‐Neu5Ac hydroxylase from the starfish A. rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength. In order to shed light on the structural basis for these differences, the primary structure of CMP‐Neu5Ac hydroxylase from A. rubens has been determined by PCR and cDNA‐cloning techniques, using initial sequence information from the mouse enzyme. The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa. The deduced amino‐acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C‐terminus was some 60 residues longer. This extension consists of a terminal hydrophobic region, which may mediate membrane‐binding, and a preceeding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of active CMP‐Neu5Ac hydroxylase in E. coli at low temperatures.
Targeted disruption of a pupal hemocyte protein of Sarcophaga by RNA interference
Authors: Nishikawa Takeshi , Natori Shunji
Published: March 2003

Previously, we purified a transmembrane protein with a molecular mass of 120 kDa (p120) that is exclusively expressed in pupal hemocytes of Sarcophaga. In this study, we demonstrated that double‐stranded RNA (dsRNA) injected into the larval body cavity effectively inhibited the expression of p120 in pupal hemocytes. Thus, RNA interference (RNAi) was found to be a useful technique for creating pupal hemocytes with a loss‐of‐function of a specific protein. The p120‐less pupal hemocytes generated by RNAi were found to have lost the ability to take up acetylated low density lipoprotein, indicating that p120 is a scavenger receptor specifically expressed on the surface of pupal hemocytes.
Isolation and characterization of a novel type of neurotoxic peptide from the venom of the South African scorpion Parabuthus transvaalicus (Buthidae)
Authors: Inceoglu Bora , Lango Jozsef , Wu Jennifer , Hawkins Pauline , Southern James , Hammock Bruce D.
Published: March 2003

The venom of the South African scorpion Parabuthus transvaalicus was characterized using a combination of mass spectrometry and RP‐HPLC separation and bioassays. The crude venom was initially separated into 10 fractions. A novel, moderately toxic but very high abundance peptide (birtoxin) of 58 amino‐acid residues was isolated, identified and characterized. Each purification step was followed by bioassays and mass spectroscopy. First a C4 RP‐HPLC column was used, then a C18 RP Microbore column purification resulted in > 95% purity in the case of birtoxin from a starting material of 230 µg of crude venom. About 12–14% of the D214 absorbance of the total venom as observed after the first chromatography step was composed of birtoxin. This peptide was lethal to mice at low microgram quantities and it induced serious symptoms including tremors, which lasted up to 24 h post injection, at submicrogram amounts. At least seven other fractions that showed different activities including one fraction with specificity against blowfly larvae were identified. Identification of potent components is an important step in designing and obtaining effective anti‐venom. Antibodies raised against the critical toxic components have the potential to block the toxic effects and reduce the pain associated with the scorpion envenomation. The discovery of birtoxin, a bioactive long chain neurotoxin peptide with only three disulfide bridges, offers new insight into understanding the role of conserved disulfide bridges with respect to scorpion toxin structure and function.
A Drosophila MBD family member is a transcriptional corepressor associated with specific genes
Authors: Ballestar Esteban , Pile Lori A. , Wassarman David A. , Wolffe Alan P. , Wade Paul A.
Published: March 2003

DNA methylation in Drosophila melanogaster is restricted temporally during development and occurs at a significantly lower frequency than in mammals. Thus, the regulatory functions, if any, of this form of DNA modification in Drosophila are unclear. However, the presence of homologs of vertebrate methyl‐CpG‐binding proteins implies functional consequences for DNA methylation in flies. This work describes the properties of dMBD‐like, a Drosophila homolog of vertebrate MBD2 and MBD3. dMBD‐like and dMBD‐likeδ (a splice variant) failed to bind model methylated DNA probes, inconsistent with their function as mediators of methyl CpG‐directed transcriptional repression. However, the MBD‐like proteins exhibit transcriptional and biochemical properties consistent with roles as components of a histone deacetylase‐dependent corepressor complex similar to the vertebrate Mi‐2 complex. The two proteins are differentially expressed during development, suggesting functional specialization. dMBD‐like and/or dMBD‐likeδ is present at the chromocenter on larval polytene chromosomes as well as at discrete bands interspersed along the euchromatic chromosome arms, many of which are coincident with known ecdysone‐induced loci. This banding pattern suggests gene‐specific regulatory functions for dMBD‐like and the Drosophila Mi‐2 complex.
Fusogenic potential of prokaryotic membrane lipids.
Authors: Ahmad N. , Masood A. K. , Owais M.
Published: March 2003

Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral‐ and cell mediated‐immunity. The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses. In contrast, the activation of a cell‐mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells. The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids. These lipids were shown to induce strong membrane–membrane fusion as evident from resonance energy transfer and content mixing assays. Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells. Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes. Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses.
Binding of gelsolin domain 2 to actin
Authors: Renoult Celine , Blondin Laurence , Fattoum Abdellatif , Ternent Diane , Maciver Sutherland K. , Raynaud Fabrice , Benyamin Yves , Roustan Claude
Published: March 2003

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin‐binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin‐binding proteins and it has been proposed that there is a similarity in the actin‐binding interface. Gelsolin domains 1 and 4 bind G‐actin in a similar manner and compete with each other, whereas domain 2 binds F‐actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F‐actin binding of domain 2 with respect to the F‐actin severing and capping activity of the whole gelsolin molecule.
Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic β‐amylase from Calystegia sepium (hedge bindweed) rhizomes
Authors: Van Damme Els J. M. , Hu Jialiang , Barre Annick , Hause Bettina , Baggerman Geert , Rougé Pierre , Peumans Willy J.
Published: March 2003

An abundant catalytically active β‐amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium). Biochemical analysis of the purified protein, molecular modeling, and cloning of the corresponding gene indicated that this enzyme resembles previously characterized plant β‐amylases with regard to its amino‐acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the β‐amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome β‐amylase is a cytoplasmic vegetative storage protein.
Pyridinyl polythiazole class peptide antibiotic micrococcin P1, secreted by foodborne Staphylococcus equorum WS2733, is biosynthesized nonribosomally
Authors: Carnio Markus C. , Stachelhaus Torsten , Francis Kevin P. , Scherer Siegfried
Published: March 2003

Recently, foodborne Staphylococcus equorum WS2733 was isolated from a French red smear cheese on account of its strong inhibitory activity against Gram‐positive pathogens such as Listeria. The antagonistic substance was identified as macrocyclic peptide antibiotic micrococcin P1, which had previously not been reported for the genus Staphylococcus. Micrococcin P1, also a potent inhibitor of the malaria parasite Plasmodium falciparum, is structurally related to thiostrepton, thiocillins and nosiheptide. Although all of these peptide antibiotics have been known for quite a long time, their mode of biosynthesis had not been determined in detail yet. By using degenerated PCR, a gene fragment encoding a nonribosomal peptide synthetase (NRPS) could be amplified from S. equorum. The corresponding chromosomal locus was disrupted by insertional mutagenesis, and it could be shown that all mutants obtained displayed a micrococcin P1‐deficient phenotype. Sequence analysis of a coherent 2.8‐kb fragment revealed extensive homology to known NRPSs, and allowed the assignment of the domain organization ‘condensation‐adenylation‐thiolation‐condensation’; an arrangement predicted only for two loci within the presumably 14‐modular, 1.6‐MDa biosynthetic NRPS template. Biochemical characterization of the adenylation domain exhibited selectivity for the substrate amino‐acid threonine. All of these data substantiate that the macrocyclic peptide antibiotic is biosynthesized nonribosomally, and provide the basis for the characterization of the entire biosynthetic gene cluster. The biosynthetic machinery of micrococcin will serve as a model system for structurally related, pharmacologically important pyridinyl polythiazole class peptide antibiotics. Furthermore, this knowledge will enable the manipulation of its NRPS template, which in turn may grant the targeted engineering of even more potent anti‐listerial and anti‐malaria drugs.
Activation and stabilization of α‐chymotrypsin by cationic additives
Authors: Spreti Nicoletta , Di Profio Pietro , Marte Luisa , Bufali Simone , Brinchi Lucia , Savelli Gianfranco
Published: March 2003

α‐Chymotrypsin activity was tested with N‐glutaryl‐l‐phenylalanine p‐nitroanilide (GPNA) in aqueous media in the presence of synthetic surfactants, which differ in the flexibility of their bulky head groups. Superactivity can be ascribed to the presence of the tributylammonium residue on the surfactant head group, as in p‐octyloxybenzyltributylammonium bromide (pOOTBABr), while in the presence of a more rigid moiety, i.e. a cyclic one, no activation was found. A nonmicellizable quaternary ammonium salt, the tetrabutylammonium bromide (TBABr), which has a head group structure very similar to pOOTBABr, not only induces a remarkable superactivation, at a concentration 80‐fold higher than pOOTBABr, but also allows the enzyme to retain a high residual activity for long periods of time. The presence of a lipophilic chain, which by interacting with apolar residues on the enzyme surface, probably penetrates into hydrophobic pockets of the protein and causes a rapid inactivation.

In 0.4 m TBABr, a 20‐fold increase both in kcat and Km values, with respect to buffer alone, was found. The increase of Km could be attributed either to a true decrease in affinity between enzyme and substrate or alternatively to the presence of TBA+ ions near the catalytic region. They could interact with protein residues around the active site and bind to negatively charged GPNA molecules, lowering the local substrate concentration.

Spectroscopic experiments (CD and fluorescence) show minor changes of protein conformation in 0.4 m TBABr, while at 1 m a strong modification of both spectra was observed.
Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi
Authors: Judice Wagner A. S. , Cezari Maria Helena S. , Lima Ana Paula C. A. , Scharfstein Julio , Chagas Jair Ribeiro , Tersariol Ivarne L. S. , Juliano Maria A. , Juliano Luiz
Published: March 2003

The Trypanosoma cruzi cysteine protease cruzipain contains a 130‐amino‐acid C‐terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose‐type, hybrid monoantennary‐type or complex biantenary‐type oligosacharide chains at Asn255 of the C‐terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2′ subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz‐KLRFSKQ‐EDDnp (Abz, ortho‐aminobenzoic acid; EDDnp, N‐(2,4‐dinitrophenyl)‐ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH–activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k−1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz‐KLRFSKQ‐EDDnp determining the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci.9, 1589–1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E−1, which are significantly higher for cruzipain. The corresponding ΔS1 and ΔS−1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C‐terminal extension and/or glycosylation state of cruzipain.
Structure of proline 3‐hydroxylase
Authors: Clifton Ian J. , Hsueh Li‐Ching , Baldwin Jack E. , Harlos Karl , Schofield Christopher J.
Published: March 2003

Iron (II)/2‐oxoglutarate (2‐OG)‐dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post‐translational modification of collagen. 2‐OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2‐OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3‐hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2‐OG oxygenase catalysing oxidation of a free α‐amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3Å resolution, R=20.2%, Rfree=25.3%) and that complexed to iron (II) (to 2.4Å resolution, R=19.8%, Rfree=22.6%). The structure contains conserved motifs present in other 2‐OG oxygenases including a ‘jelly roll’β strand core and residues binding iron and 2‐oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2‐OG oxygenases in possessing a discrete C‐terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2‐OG turnover.
Structures of two O‐chain polysaccharides of Citrobacter gillenii O9a,9b lipopolysaccharide
Authors: Lipiński Tomasz , Zatonsky George V. , Kocharova Nina A. , Jaquinod Michel , Forest Eric , Shashkov Alexander S. , Gamian Andrzej , Knirel Yuriy A.
Published: March 2003

Mild acid degradation of the lipopolysaccharide of Citro‐ bacter gillenii O9a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4‐acetamido‐4,6‐dideoxy‐d‐mannose (d‐Rha4NAc, N‐acetyl‐d‐perosamine). PS was studied by methylation analysis␣and 1H‐NMR and 13C‐NMR spectroscopy, using two‐dimensional 1H,1H COSY, TOCSY, NOESY, and H‐detected 1H,13C heteronuclear correlation experiments. It was␣found that PS includes two structurally different polysaccharides: an α1→2‐linked homopolymer of N‐acetyl‐d‐perosamine [→2)‐α‐d‐Rhap4NAc‐(1→, PS2] and a␣polysaccharide composed of tetrasaccharide repeating units (PS1) with the␣following structure:

→3)‐α‐d‐Rhap4NAc‐(1→2)‐α‐d‐Rhap4NAc‐(1→2)‐α‐d‐Rhap4NAc‐(1→3)‐α‐d‐Rhap4N Ac2Ac‐(1→

where the degree of O‐acetylation of a 3‐substituted Rha4NAc residue at position 2 is ≈ 70%.  

PS could be fractionated into PS1 and PS2 by gel‐permeation chromatography on TSK HW‐50S. Matrix‐assisted laser desorption ionization MS data indicate sequential chain elongation of both PS1 and PS2 by a single sugar unit, with O‐acetylation in PS1 beginning at a certain chain length. Anti‐(C. gillenii O9a,9b) serum reacted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a structurally related O‐chain polysaccharide were reactive.
Acceleration of granulocyte colony‐stimulating factor‐induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinase
Authors: Omura Tomomi , Sakai Hiroshi , Murakami Hiroshi
Published: March 2003

Stimulation with granulocyte colony‐stimulating factor (G‐CSF) induces myeloid precursor cells to differentiate into neutrophils, and tyrosine phosphorylation of certain cellular proteins is crucial to this process. However, the signaling pathways for neutrophil differentiation are still obscure. As the Src‐like tyrosine kinase, Lyn, has been reported to play a role in G‐CSF‐induced proliferation in avian lymphoid cells, we examined its involvement in G‐CSF‐induced signal transduction in mammalian cells. Expression plasmids for wild‐type Lyn (Lyn) and kinase‐negative Lyn (LynKN) were introduced into a murine granulocyte precursor cell line, GM‐I62M, that can respond to G‐CSF with neutrophil differentiation, and cell lines that overexpressed these molecules (GM‐Lyn, GM‐LynKN) were established. Upon G‐CSF stimulation, both the GM‐Lyn and GM‐LynKN cells began to differentiate into neutrophils, showing early morphological changes within a few days, much more rapidly than did the parental cells, which started to exhibit nuclear lobulation about 10 days after the cells were transferred to G‐CSF‐containing medium. However, the time course of expression of the myeloperoxidase gene, another neutrophil differentiation marker, was not affected by the overexpression of Lyn or LynKN. Therefore, in normal cells, protein interactions with Lyn, but not its kinase activity, are important for the induction of G‐CSF‐induced neutrophilic nuclear lobulation in mammalian granulopoiesis.
Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt
Authors: Kazantzis Athanasios , Waldner Michaela , Taylor John W. , Kapurniotu Aphrodite
Published: March 2003

Calcitonin (Ct) is a 32‐residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our previous studies have led to potent, side‐chain lactam‐bridged human Ct (hCt) analogues [Kapurniotu, A. Kayed, R., Taylor, J.W. & Voelter W. (1999) Eur. J. Biochem.265, 606–618; Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem.38, 836–847]. We have hypothesized that a possibly type I β turn/β sheet conformation in the region 17–21 may play an important role in hCt bioactivity. To investigate this hypothesis, analogues of the potent hCt agonist cyclo17,21‐[Asp17,Lys21]hCt (1) bearing type I (and II′) or II β turn‐promoting substituents at positions 18 and 19 were designed, synthesized and their solution conformations, human Ct receptor binding affinities and in vivo hypocalcemic potencies were assessed. The novel analogues include cyclo17,21‐[Asp17,d‐Phe19, Lys21]hCt (2), cyclo17,21‐[Asp17,Aib18,Lys21]hCt (3), cyclo17,21‐[Asp17,d‐Lys18,Lys21]hCt (4), corresponding partial sequence peptides containing the lactam‐bridged region 16–22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas analogues 2–4 had similar receptor affinities to hCt. In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1, whereas 2 completely lost the high potency of 1, suggesting that type I (and II′) β turn‐promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analogues 1–4 were markedly β sheet‐stabilized compared to hCt and indicated the presence of distinct β turn conformeric populations in each of the analogues. Unexpectedly, the d‐amino acid‐ or Aib‐containing cyclic analogues 2–4 but not 1 or hCt self‐associated into SDS denaturation‐stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17–21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state of hCt. These results may assist to delineate the structure‐function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone‐disorder‐related diseases.
Intermonomer flexibility of Ca‐ and Mg‐actin filaments at different pH values
Authors: Hild Gábor , Nyitrai Miklós , Somogyi Béla
Published: March 2003

The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca‐actin and Mg‐actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The␣protomers were labeled on Cys374 with donor [N‐(((iodoacetyl)amino)ethyl)‐5‐naphthylamine‐1‐sulfonate; IAEDANS] or acceptor [5‐(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg‐F‐actin while this difference was absent in the case of Ca‐F‐actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg‐F‐actin compared to the Ca‐F‐actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg‐ and Ca‐actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the␣magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca‐actin filaments.
Kinetic study of sn‐glycerol‐1‐phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1
Authors: Han Jin‐Suk , Kosugi Yoshitsugu , Ishida Hiroyasu , Ishikawa Kazuhiko
Published: March 2003

A gene having high sequence homology (45–49%) with the glycerol‐1‐phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94–96 °C at near neutral pH. This enzyme was subjected to two‐substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H‐ dependent dihydroxyacetone phosphate reduction and NAD+‐dependent␣glycerol‐1‐phosphate (Gro1P) oxidation. NADP+‐dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol‐ 1‐phosphate dehydrogenase from A. pernix follows a ordered bi–bi mechanism.
A novel gain‐of‐function mutation of the integrin α2 VWFA domain
Authors: Aquilina Alexis , Korda Michelle , Bergelson Jeffrey M. , Humphries Martin J. , Farndale Richard W. , Tuckwell Danny
Published: March 2003

Integrin α2β1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of α2β1 has been localized to the α2 von Willebrand Factor type A (VWFA) domain (A‐domain or I‐domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of α2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain‐of‐function mutation. Recombinant α2‐E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild‐type. Side chain hydrophobicity was important for the gain‐of‐function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as α2‐E318W VWFA domain differed from the wild‐type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain‐of‐function effect was not restricted to binding to collagen I as α2‐E318W also showed elevated binding to collagen IV, collagen I C‐propeptide, laminin and E‐cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating α2 VWFA domain–ligand binding and may be involved in the conformational changes associated with its regulation.
Synthesis and turn‐over of the replicative Cdc6 protein during the HeLa cell cycle
Authors: Biermann Esther , Baack Martina , Kreitz Sandra , Knippers Rolf
Published: March 2003

The human replication protein Cdc6p is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle. However, the amounts of Cdc6p on chromatin remain high during S phase implying either that displaced Cdc6p can rebind to chromatin, or that Cdc6p is synthesized de novo. We have performed metabolic labeling experiments and determined that [35S]methionine is incorporated into Cdc6p at similar rates during the G1 phase and the S phase of the cell cycle. Newly synthesized Cdc6p associates with chromatin. Pulse–chase experiments show that chromatin‐bound newly synthesized Cdc6p has a half life of 2–4 h. The results indicate that, once bound to chromatin, pulse‐labeled new Cdc6p behaves just as old Cdc6p: it dissociates and eventually disappears from the nucleus. The data suggest a surprisingly dynamic behaviour of Cdc6p in the HeLa cell cycle.
Regulation of stress‐activated protein kinase signaling pathways by protein phosphatases
Authors: Tamura Shinri , Hanada Masahito , Ohnishi Motoko , Katsura Koji , Sasaki Masato , Kobayashi Takayasu
Published: March 2003

Stress‐activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.
An ELISA for recombinant Lepidoglyphus destructor, Lep d 2, and the monitoring of exposure to dust mite allergens in farming households
Authors: Parvaneh S. , Johansson E. , Elfman L. H. M. , Van Hage‐Hamsten M.
Published: March 2003

Background Exposure to indoor allergens, such as dust mites, has been recognized as a risk factor for sensitization and symptoms.

Objective To develop a two‐site ELISA for the determination of Lep d 2 in the reservoir, to measure dust mite allergen exposure (Lep d 2, Der p 1, Der f 1 and Der 2) in farm households, and to investigate whether exposure to these allergens is associated with sensitization, asthma and rhinoconjunctivitis.

Methods Monoclonal antibodies to recombinant (r)Lep d 2 were produced with standard hybridoma technique. Dust samples from 393 households were analysed for allergen content by two‐site ELISA methods.

Results A two‐site Lep d 2 ELISA was developed with a detection limit of 0.09 µg/g. The assay was highly reproducible and levels of Lep d 2 showed a strong correlation with the number of Lepidoglyphus mites (rs: 0.7; P = 0.0002). Lep d 2 was detected in 20% of the homes; levels ranged from 0.09 to 1.7 µg/g of dust. Der p 1 was recorded in 59% of the samples, ranging from 0.055 to 139 µg/g, and Der f 1 and Der 2 in 40% and 50% of the samples, ranging from 0.055 to 24.5 µg/g and 24.3 µg/g, respectively. Dermatophagoides allergens were significantly higher in mattresses than in carpets (P < 0.0001), but this difference was not observed with Lep d 2. A strong relationship between immunoglobulin (Ig)E to rLep d 2 and asthma (OR = 10.4) and rhinoconjunctivitis (OR = 7.5) was seen. Furthermore, sensitization to D. pteronyssinus was significantly associated with asthma (OR: 13.7) and rhinoconjunctivitis (OR: 5.7).

Conclusion When assessing mite allergen exposure in rural homes, not only the Der p 1, Der f 1 and Der 2 allergens, but also the Lep d 2 allergen should be taken into consideration.
Empirically simulated study to compare and validate sampling methods used in aerial surveys of wildlife populations
Authors: Khaemba Wilson Mwale , Stein Alfred , Rasch Dieter , De Leeuw Jan , Georgiadis Nick
Published: March 2003

This paper compares the distribution, sampling and estimation of abundance for two animal species in an African ecosystem by means of an intensive simulation of the sampling process under a geographical information system (GIS) environment. It focuses on systematic and random sampling designs, commonly used in wildlife surveys, comparing their performance to an adaptive design at three increasing sampling intensities, using the root mean square errors (RMSE). It further assesses the impact of sampling designs and intensities on estimates of population parameters. The simulation is based on data collected during a prior survey, in which geographical locations of all observed animals were recorded. This provides more detailed data than that usually available from transect surveys. The results show precision of estimates to increase with increasing sampling intensity, while no significant differences are observed between estimates obtained under random and systematic designs. An increase in precision is observed for the adaptive design, thereby validating the use of this design for sampling clustered populations. The study illustrates the benefits of combining statistical methods with GIS techniques to increase insight into wildlife population dynamics.
Feature
Authors: Ford Trevor D. , Quirk David , Burnett David
Published: March 2003

The last decade has seen a complete revision of the sedimentary environment, stratigraphic sequence and structural history of the early Ordovician Manx ‘Slate’ Group of the Isle of Man.