bluuebunny/crossref_metadata_2025 huggingface dataset:
Insect silk contains both a Kunitz‐type and a unique Kazal‐type proteinase inhibitor
Authors: Nirmala Xavier , Kodrík Dalibor , Žurovec Michal , Sehnal František
Published: March 2003

Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteins, and contains a few small peptides of hitherto unknown functions. We demonstrate that two of these peptides inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These ‘silk proteinase inhibitors’ 1 and 2 (SPI 1 and 2) are produced in the middle section of the silk‐secreting glands prior to cocoon spinning and their production is controlled at transcription level. The full length cDNA of pre‐SPI 1 contains 443 nucleotides and encodes a peptide of 76 amino‐acid residues, of which 20 make up a signal sequence. The mature SPI 1 (6056.7 Da, 56 residues) is a typical thermostable Kunitz‐type proteinase inhibitor with Arg in P1 position. The cDNA of pre‐SPI 2 consists of 260 nucleotides and yields a putative secretory peptide of 58 amino‐acid residues. The functional SPI 2 (3993 Da, 36 residues) is a single‐domain Kazal‐type proteinase inhibitor with unique structural features: free segment of the N‐terminus is reduced to a single amino‐acid residue, lack of CysI and CysV precludes formation of the A‐ring and provides increased flexibility to the C‐ring, and absence of several residues around the normal position of CysV shortens and changes the α helix segment of the protein. The structure reveals that the length and arrangement of the B‐ring, including exposure of the P1 residue, and the position of the C‐terminus relative to the B‐loop, are essential for the activity of the Kazal‐type inhibitors.
Pressure‐exploration of the 33‐kDa protein from the spinach photosystem II particle
Authors: Ruan Kangcheng , Xu Chunhe , Yu Yong , Li Jiong , Lange Reinhard , Bec Nicole , Balny Claude
Published: March 2003

The 33‐kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high‐pressure protein‐unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 °C and pH 6, the 33‐kDa protein shows a reversible two‐state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (ΔGo) and in volume (ΔV) for the transition at pH 6.0 and 20 °C are −14.6 kJ·mol−1 and −120 mL·mol−1, respectively, in which the ΔGo value is consistent with that obtained by chemical denaturation. We found that pressure‐induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of ΔGo (only a minor variation was observed for the ΔV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in ΔV without any significant changes in ΔGo. It was also found that NaCl significantly protects the protein from pressure‐induced unfolding. In the presence of 1 m NaCl, the pressure needed to induce the half‐unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of ΔV is significantly reduced whilst that of ΔGo remains as before. The unfolding‐refolding kinetics of the protein has also been studied by pressure‐jump, in which it was revealed that both reactions are a two‐state transition process with a relatively slow relaxation time of about 102 s.
Palmitate uptake by neonatal rat myocytes and hepatocytes
Authors: Elmadhoun Bassam M. , Wang Gu Qi , Kirshenbaum Lorrie A. , Burczynski Frank J.
Published: March 2003

The role of extracellular binding proteins in the rate of [3H]palmitate uptake by neonatal cardiac myocytes and hepatocytes was investigated using a model‐independent approach. Binding proteins used in this study included α1‐acid glycoprotein [isoelectric point (pI) ≈ 2.7], conalbumin (pI ≈ 6.4), lysozyme (pI ≈ 11.0), albumin (pI ≈ 4.9), and albumin which had been modified to yield proteins with pI values of 3.5, 4.7, 7.5 and 8.6. All uptake studies were conducted at similar unbound ligand fractions. There was a linear relationship between the rate of neonatal hepatocyte [3H]palmitate clearance and protein pI (r2 = 0.98). In contrast, there was an overall poor relationship between neonatal cardiac myocyte [3H]palmitate‐clearance rate and protein pI (r2 = 0.48). However, the relationship improved when the data on [3H]palmitate‐clearance were analyzed using only the modified albumins. The study indicates that an ionic interaction between extracellular proteins and the hepatocyte surface enhances the overall uptake of [3H]palmitate. This interaction may be limited to albumin for neonatal cardiac myocytes.
Structure of an acidic exopolysaccharide produced by the diazotrophic endophytic bacterium Burkholderia brasiliensis
Authors: Mattos Katherine A. , Jones Christopher , Heise Norton , Previato José O. , Mendonça‐Previato Lúcia
Published: March 2003

The structure of an exopolysaccharide (EPS) produced by Burkholderia brasiliensis, a diazotrophic endophytic organism originally isolated from rice roots, has been determined. The bacterium was grown in a synthetic medium, containing mannitol and glutamate, which favours the expression of two anionic EPSs, which were separated by anion‐exchange chromatography. The structure of the repeat unit of EPS A, eluted at higher ionic strength, was determined by a combination of methylation analysis, partial hydrolysis, chemical degradations, and NMR spectroscopic studies, and shown to be the linear O‐acetylated pentasaccharide: →4)‐α‐d‐Glcp‐(1→2)‐α‐l‐Rhap‐(1→4)‐α‐d‐GlcpA‐(1→3)‐β‐l‐Rhap[2OAc]‐(1→4)‐β‐d‐Glcp‐(1→
Biochemical characterization and surfactant properties of horse allergens
Authors: Botros Hany Goubran , Poncet Pascal , Rabillon Jacques , Fontaine Thierry , Laval Jean‐Marc , David Bernard
Published: March 2003

A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri‐antennary tri‐sialylated N‐linked glycan. Linkages of terminal N‐acetylneuraminic acid to galactose were: α‐(2→6) in Equ c 4, and both α‐(2→3) and α‐(2→6) in Equ c 1. Oligosaccharide portions of Equ c 1 or Equ c 4 were barely involved in IgE‐immunoreactivity. Partial N‐terminal sequence of Equ c 4 shares a significant sequence homology with the rat submandibular gland protein A. No matching was found for two internal peptides of Equ c 5. Surfactant properties of horse allergens as well as other proteins were investigated. In contrast to Equ c 2 and Equ c 3, solutions of Equ c 1, Equ c 4 and Equ c 5 significantly lowered the surface tension. Relationship between a property such as this, involving oriented hydrophobic patches of a molecule and allergenicity, is addressed.
C‐Terminal region of human NAD+‐dependent 15‐hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates
Authors: Zhou Huiping , Yan Fengxiang , Tai Hsin‐Hsiung
Published: March 2003

NAD+‐dependent 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15‐keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15‐PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C‐terminus. The involvement of the C‐terminal region in catalytic activity was examined by studies on C‐terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C‐terminal end of human 15‐PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C‐terminal 14 amino acids of human 15‐PGDH were replaced by the C‐terminal 14 amino acids of rat 15‐PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild‐type enzymes. This indicates that the catalytic effectiveness of human 15‐PGDH decreases as the C‐terminal region is gradually removed or replaced by rat sequences. The C‐terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.
Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus
Authors: Ogasahara Kyoko , Khechinashvili Nikolay N. , Nakamura Mamoru , Yoshimoto Tadashi , Yutani Katsuhide
Published: March 2003

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90–95 °C and its activity was higher in the temperature range 30–100 °C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 °C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.
Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH
Authors: Salvati Luca , Mattu Marco , Polticelli Fabio , Tiberi Federica , Gradoni Luigi , Venturini Giorgio , Bolognesi Martino , Ascenzi Paolo
Published: March 2003

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite–host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain‐like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and Ki for the cruzipain‐catalysed hydrolysis of N‐α‐benzyloxycarbonyl‐l‐phenylalanyl‐l‐arginine‐(7‐amino‐4‐methylcoumarin) (Z‐Phe‐Arg‐AMC) and azocasein were determined between 10.0 °C and 40.0 °C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and Ki were temperature‐dependent and pH‐dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logKi increased linearly with respect to T −1. Values of Vmax and Vmax/Km were affected by the acid‐base equilibrium of one temperature‐independent ionizing group (i.e. pKunl′ = pKlig′ = 5.7 ± 0.1, at 25.0 °C). Moreover, values of Ki were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pKunl″ = 5.7 ± 0.1 to pKlig″ = 6.1 ± 0.1, at 25.0 °C) upon Z‐Phe‐Arg‐AMC binding. Values of logKunl′, logKlig′, and logKlig″ were temperature‐independent. Conversely, values of logKunl″ were linearly dependent on T −1. As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.
Purification, cDNA cloning and characterization of the vascular apoptosis‐inducing protein, HV1, from Trimeresurus flavoviridis
Authors: Masuda Shinako , Hayashi Hiroshi , Atoda Hideko , Morita Takashi , Araki Satohiko
Published: March 2003

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis‐inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis‐inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino‐acid residues. The amino‐acid sequence predicted from the cDNA was highly homologous to VAP1. The amino‐acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin‐like domain and a cysteine‐rich domain. In the disintegrin‐like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.
Deduced amino‐acid sequence of a calcium‐free α‐amylase from a strain of Bacillus
Authors: Hagihara Hiroshi , Hayashi Yasuhiro , Endo Keiji , Igarashi Kazuaki , Ozawa Tadahiro , Kawai Shuji , Ozaki Katsuya , Ito Susumu
Published: March 2003

Alkaline α‐amylase (AmyK38) from the alkaliphilic Bacillus sp. strain KSM‐K38 is a unique enzyme in that it is highly chelator‐resistant and oxidatively stable [Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa‐Kitayama, K., Ozaki, K., Kawai, S. & Ito, S. (2001) Appl. Environ. Microbiol.67, 1744–1750]. This enzyme was found to contain no Ca and require Na (or monovalent cations) for manifestation of activity. The nucleotide sequence of the gene for the novel enzyme was determined, and it harbored an ORF of 1503 bp encoding the enzyme of 501 amino acids, including a 21‐amino‐acid signal peptide. The deduced amino‐acid sequence of the mature enzyme (55 097 Da) showed moderate homology to those of α‐amylases from Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, with ≈ 63% identity. A methionine residue, which is conserved and susceptible to chemical oxidation, was replaced with leucine in AmyK38. Moreover, many conserved residues that are crucial ligands for Ca were replaced with other amino acids, thereby leading to loss of the Ca coordination geometries. By building a molecular model, we showed the calcium‐independent, oxidatively stable active‐site topology and structural integrity of AmyK38.
The N‐terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts
Authors: Inoue Kentaro , Demel Rudy , de Kruijff Ben , Keegstra Kenneth
Published: March 2003

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N‐terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino‐acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein‐mediated system, and purified it by reverse‐phase HPLC. Its identity was confirmed by N‐terminal amino‐acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono‐ and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5‐bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5‐bisphosphate carboxylase/oxygenase.
Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia
Authors: Dallinger Reinhard , Wang Yunjuan , Berger Burkhard , Mackay Elaine A. , Kägi Jeremias H. R.
Published: March 2003

The Cd‐sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd‐containing MTs the occurrence of a prominent Cd‐mercaptide‐specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd‐mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal‐free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3‐to‐1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd‐bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differeed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.
Structure and characterization of the glycan moiety of L‐amino‐acid oxidase from the Malayan pit viper Calloselasma rhodostoma
Authors: Geyer Armin , Fitzpatrick Teresa B. , Pawelek Peter D. , Kitzing Karina , Vrielink Alice , Ghisla Sandro , Macheroux Peter
Published: March 2003

Ophidian l‐amino‐acid oxidase (l‐amino‐acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD‐dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian l‐amino‐acid oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of l‐amino‐acid oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogenous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI‐TOF mass spectrometry the glycan is identified as a bis‐sialylated, biantennary, core‐fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme.
Identification of protein‐coding genes in the genome of Vibrio cholerae with more than 98% accuracy using occurrence frequencies of single nucleotides
Authors: Wang Ju , Zhang Chun‐Ting
Published: March 2003

The published sequence of the Vibrio cholerae genome indicates that, in addition to the genes that encode proteins of known and unknown function, there are 1577 ORFs identified as conserved hypothetical or hypothetical gene candidates. Because the annotation is not 100% accurate, it is not known which of the 1577 ORFs are true protein‐coding genes. In this paper, an algorithm based on the Z curve method, with sensitivity, specificity and accuracy greater than 98%, is used to solve this problem. Twenty‐fold cross‐validation tests show that the accuracy of the algorithm is 98.8%. A detailed discussion of the mechanism of the algorithm is also presented. It was found that 172 of the 1577 ORFs are unlikely to be protein‐coding genes. The number of protein‐coding genes in the V. cholerae genome was re‐estimated and found to be ≈ 3716. This result should be of use in microarray analysis of gene expression in the genome, because the cost of preparing chips may be somewhat decreased. A computer program was written to calculate a coding score called VCZ for gene identification in the genome. Coding/noncoding is simply determined by VCZ > 0/VCZ < 0. The program is freely available on request for academic use.
Structure of the O‐specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33
Authors: Zych Krystyna , Toukach Filip V. , Arbatsky Nikolay P. , Kołodziejska Katarzyna , Senchenkova Sof'ya N. , Shashkov Alexander S. , Knirel Yuriy A. , Sidorczyk Zygmunt
Published: March 2003

The acidic O‐specific polysaccharide chain (O‐antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H‐NMR and 13C‐NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H‐detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain d‐ribitol 5‐phosphate (d‐Rib‐ol‐5‐P) and ethanolamine phosphate (Etn‐P) and has the following structure

This structure is identical with that of the O‐polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O‐antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross‐reactivity of P. mirabilis D52 O‐antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O‐polysaccharides. The role of charged groups, Rib‐ol‐5‐P and Etn‐P in the immunospecificity is discussed.
Localization of epitopes for monoclonal antibodies to urokinase‐type plasminogen activator
Authors: Petersen Helle H. , Hansen Martin , Schousboe Susanne L. , Andreasen Peter A.
Published: March 2003

We localized the epitopes for several murine mAbs to human urokinase‐type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so‐called 37‐loop and 60‐loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor‐1 (PAI‐1). Besides inhibiting uPA‐catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI‐1, decreasing the second‐order rate constant 15‐ to 6500‐fold. There was no correlation between the relative effects of the 37‐loop and 60‐loop substitutions on the second‐order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA–PAI‐1 reaction, but rather by steric hindrance of the access of PAI‐1 to the active site. The affinity of the SPD antibodies for the uPA–PAI‐1 complex was only slightly lower than that for free uPA, indicating that the 37‐loop and 60‐loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA–polyanion affinity after substitution of the three Arg residues.
Functional analysis of polar amino‐acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger
Authors: Murtazina Rakhilya , Booth Brenda J. , Bullis Bonnie L. , Singh Dyal N. , Fliegel Larry
Published: March 2003

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site‐specific mutagenesis was used to examine the functional role of conserved, polar amino‐acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino‐acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262→Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262→Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side‐chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino‐acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.
Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two‐dimensional gel electrophoresis and peptide mass fingerprinting
Authors: Veith Paul D. , Talbo Gert H. , Slakeski Nada , Reynolds Eric C.
Published: March 2003

Porphyromonas gingivalis is a Gram‐negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40–41 kDa. The proteins in the train of spots were digested in‐gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N‐terminal and C‐terminal fragment of both proteins. The N‐terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino‐acid sequence of these N‐terminal fragments in each 2D gel spot was verified suggesting lack of post‐translational modification. Re‐subjecting a single N‐terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34‐ to 35‐kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70‐kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA‐like porins of other Gram‐negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.
Allosteric phenomena in cytochrome P450‐catalyzed monooxygenations
Authors: Hlavica Peter , Lewis David F. V.
Published: March 2003

Allosteric regulation of monooxygenase activity is shown to occur with diverse cytochrome P450 isoforms and is characterized by kinetic patterns deviating from the Michaelis–Menten model. Homotropic and heterotropic phenomena are encountered in both substrate activation and productive coupling of the electron donors NADPH–cytochrome P450 reductase and cytochrome b5, and the lipid environment of the system also appears to play a role as an effector. Circumstantial analysis reveals the components of the electron transfer chain to be mutually beneficial in interactions with each other depending on the substrate used and type of cytochrome P450 operative. It is noteworthy that association of diatomic gaseous ligands may be amenable to allosteric regulation as well. Thus, dioxygen binding to cytochrome P450 displays nonhyperbolic kinetic profiles in the presence of certain substrates; the latter, together with redox proteins such as cytochrome b5, can exert efficient control of the abortive breakdown of the oxyferrous intermediates formed. Similarly, substrates may modulate the structural features of the access channel for solutes such as carbon monoxide in specific cytochrome P450 isozymes to either facilitate or impair ligand diffusion to the heme iron. The in vivo importance of allosteric regulation of enzyme activity is discussed in detail.
All or none fibrillogenesis of a prion peptide
Authors: Zou Wen‐Quan , Yang Dun‐Sheng , Fraser Paul E. , Cashman Neil R. , Chakrabartty Avijit
Published: March 2003

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino‐acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195–213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all‐or‐none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.
Specific inhibition of interleukin‐4‐dependent Stat6 activation by an intracellularly delivered peptide
Authors: Stolzenberger Sascha , Haake Markus , Duschl Albert
Published: March 2003

The transcription factor Stat6 (signal transducer and activator of transcription 6) is activated following stimulation with interleukin (IL)‐4 or IL‐13. Stat6 binds via a single SH2 domain first to tyrosine‐phosphorylated motifs in the IL‐4Rα chain, and then to another Stat6 molecule, which results in the formation of active dimers. We show here that a peptide derived from the Stat6‐binding region of IL‐4Rα (Stat6BP) is an effective inhibitor when it is delivered into cells by coupling with a membrane‐permeable peptide. Stat6BP completely inhibited IL‐4 dependent phosphorylation of Stat6, as well as basal and IL‐4 stimulated transcription from a reporter gene construct with a Stat6‐dependent promoter, while IL‐3 and IL‐4 dependent phosphorylation of Stat5 was not affected. The inhibitory effect of Stat6BP was transient, but could be prolonged by treating the cells with the phosphatase inhibitor pervanadate.
DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene‐expression and metabolic regulation of both topoisomerase I and DNA gyrase
Authors: Snoep Jacky L. , van der Weijden Coen C. , Andersen Heidi W. , Westerhoff Hans V. , Jensen Peter Ruhdal
Published: March 2003

DNA of prokaryotes is in a nonequilibrium structural state, characterized as ‘active’ DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell34, 105–113]. We here report on a new method for quantifying homeostatic control of the high‐energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem.266, 865–877]. Of the two regulatory mechanisms via which homeostasis is␣conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild‐type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).
Binding of N‐acetyl‐N ′‐β‐d‐glucopyranosyl urea and N‐benzoyl‐N ′‐β‐d‐glucopyranosyl urea to glycogen phosphorylase b
Authors: Oikonomakos Nikos G. , Kosmopoulou Magda , Zographos Spyros E. , Leonidas Demetres D. , Chrysina Evangelia D. , Somsák László , Nagy Veronika , Praly Jean‐Pierre , Docsa Tibor , Tóth Béla , Gergely Pál
Published: March 2003

Two substituted ureas of β‐d‐glucose, N‐acetyl‐N′‐β‐d‐glucopyranosyl urea (Acurea) and N‐benzoyl‐N′‐β‐d‐glucopyranosyl urea (Bzurea), have been identified as inhibitors of glycogen phosphorylase, a potential target for therapeutic intervention in type 2 diabetes. To elucidate the structural basis of inhibition, we determined the structure of muscle glycogen phosphorylase b (GPb) complexed with the two compounds at 2.0 Å and 1.8 Å resolution, respectively. The structure of the GPb–Acurea complex reveals that the inhibitor can be accommodated in the catalytic site of T‐state GPb with very little change in the tertiary structure. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the GPb–glucose complex, while the acetyl urea moiety is in a favourable electrostatic environment and makes additional polar contacts with the protein. The structure of the GPb–Bzurea complex shows that Bzurea binds tightly at the catalytic site and induces substantial conformational changes in the vicinity of the catalytic site. In particular, the loop of the polypeptide chain containing residues 282–287 shifts 1.3–3.7 Å (Cα atoms) to accommodate Bzurea. Bzurea can also occupy the new allosteric site, some 33 Å from the catalytic site, which is currently the target for the design of antidiabetic drugs.
Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori
Authors: Srinivasan Lakshmi , Gopinathan Karumathil P.
Published: March 2003

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori, resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51‐kDa subunit could be specifically crosslinked to the B box of

. Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification, the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.
How Rearing History Affects Alarm‐call Responses of Belding’s Ground Squirrels (Spermophilus beldingi, Sciuridae)
Authors: Mateo Jill M. , Holmes Warren G.
Published: March 2003

Juvenile, but not adult, Belding’s ground squirrels (Spermophilus beldingi) exhibit markedly different responses to alarm calls as a function of their environment. Compared with same‐aged, free‐living juveniles, captive juveniles (housed in large outdoor enclosures) are more likely to respond to playbacks, to exhibit more exaggerated initial responses (e.g. enter a burrow vs. freeze) and to remain alert longer following playbacks of alarm and non‐alarm calls. Two studies were conducted to identify the factors contributing to these response differences. Postemergent rearing environments (such as the opaque enclosure walls that limited visual and auditory stimulation in captivity, or the increased number of conspecifics and natural alarm calls that free‐living juveniles experienced) could not account for the majority of response differences between captive and free‐living juveniles (Study 1). To determine if the attenuated responses of free‐living juveniles were due to foraging pressures, we compared the behaviours of food‐provisioned captive juveniles with those of non‐provisioned captive juveniles. Although sample sizes were small, no differences were evident in the development or expression of responses as a function of foraging pressure. Next, the development of captive juveniles was compared with that of juveniles reared in the field but housed in captivity after emergence (Study 2). Differences in the response patterns of field‐reared and captive‐reared animals matched the differences reported previously, as the responses of field‐reared animals observed in captivity mirrored those of free‐living juveniles that remained in the field. Thus, the differences in alarm‐call responses originally observed between captive and free‐living juveniles are attributed to their pre‐emergent, but not post‐emergent, rearing histories.

Captive pups experienced levels of auditory, visual, tactile, and olfactory stimulation that were greater than those typically experienced by free‐living pups. The increased exposure to conspecific alarm calls may have primed captive pups to respond more often and more intensely to the auditory stimuli they heard as juveniles. Sensitivity to early rearing environments may be adaptive for young ground squirrels if it facilitates the development of antipredator behaviour patterns that are appropriate for the local predator environment (e.g. openness of habitat, frequency of predators, availability of refuges).
Agonistic Encounters in a Cricket Frog (Acris crepitans) Chorus: Behavioral Outcomes Vary with Local Competition and within the Breeding Season
Authors: Burmeister Sabrina , Konieczka Jay , Wilczynski Walter
Published: March 2003

Male cricket frogs (Acris crepitans) gather in breeding choruses and call to attract females. The call also serves to maintain an individual’s calling space. When an intruder invades a resident male’s space, the resident will display one of three behavior patterns. He will either attack the intruder, abandon calling, or tolerate the intruder’s presence and continue calling. We simulated an intruder by broadcasting a stimulus from a speaker and categorized the response of the resident male. We recorded social variables relevant to local competition among males, specifically, local caller density (within 2 m) and the amplitude of the nearest neighbor’s calls. In addition we recorded the size of the subject, the amplitude of the stimulus, and the time of night and the season. We used a multivariate approach to assess the relative importance of these variables. Local caller density and nearest neighbor call amplitude were the best predictors of a resident’s response to the intruder; higher levels of local competition resulted in more males tolerating the intruder. In addition, behavior changed over the season: males were more likely to abandon or ignore the intruder early in the season and more likely to attack later in the season. No other variables were related to the outcome of the agonistic encounter. This study suggests that the most important variables impinging on a male’s decision to fight, flee, or ignore an intruder are influences external to the individuals involved in the conflict.
Survival Benefit to Antipredator Behavior in the Amphipod Gammarus minus (Crustacea: Amphipoda) in Response to Injury‐released Chemical Cues from Conspecifics and Heterospecifics
Authors: Wisenden Brian D. , Cline Alecia , Sparkes Timothy C.
Published: March 2003

A wide range of aquatic taxa use environmental chemical cues for the assessment of predation risk. We examined whether Gammarus minus (Crustacea: Amphipoda) exhibit antipredator behavior in response to injury‐released chemicals from conspecifics or heterospecifics (Crustacea: Isopoda). We then examined whether behavioral responses to these cues conferred survival benefits to the amphipods. In the first part of this study, we tested the behavioral response of G. minus to the following treatments: 1. water containing injury‐released cues of conspecifics; 2. water containing injury‐released cues of a sympatric isopod crustacean, Lirceus fontinalis; or 3. water containing no cues (control). Relative to the control, Gammarus responded to the conspecific cue by moving to the substratum and decreasing activity. In contrast, Gammarus responded to the heterospecific cue by moving up into the water column and increasing activity. In the second part of this study, we tested if the behavioral response to these cues confers a survival benefit to Gammarus when exposed to a predator. A green sunfish (Lepomis cyanellus) was retained behind a partition in the test tanks. Two minutes after the introduction of the chemical cues in the first test, the barrier was lifted and predation events recorded. Relative to the control, the time to the first attack increased for Gammarus exposed to conspecific cues and decreased for those exposed to heterospecific cues. These data indicate that Gammarus distinguish between chemical cues from conspecific and heterospecific crustaceans, and that the antipredator response to conspecific cues confers a fitness benefit (i.e. increased survival due to increased time to the first attack).
Foraging Cost of a Long Tail Ornament: An Experiment with Sand Martin Females
Authors: Matyjasiak P. , Jab?oń P. G. ,   ski, Olejniczak I. , Boniecki P. I. , Lee S. ‐D.
Published: March 2003

The handicap hypothesis assumes that sexual ornaments impose a viability cost upon the bearers. There have been few empirical tests of this assumption. Previous studies show evidence for the cost of a tail ornament in male birds: a negative relationship between an experimentally increased tail ornament (long tail streamers) and efficiency at foraging for nestlings. However, it must be admitted, that the apparent impairing effect of an elongated tail could be a result of a decrease in male parental effort in response to an increase of female parental effort, which might have occurred in response to increased male attractiveness (differential allocation of female parental effort). In this study, the effect of differential parental expenditure was eliminated by lengthening the tail in female, rather than male, sand martins (Riparia riparia). Tail‐elongated females decreased the rate at which they fed nestlings, and captured more but smaller insects. There was no simultaneous increase of feeding rate in the males that could explain the decrease of feeding rate in the females. These results confirm the existence of a cost of a tail ornament in birds feeding in flight, as is expressed in terms of impaired flight and foraging capacity.
Does Conspecific Attraction Affect Nest Choice in Budgerigars (Melopsittacus undulatus: Psittacidae: Aves)?
Authors: Baltz Aliza P. , Clark Anne B.
Published: March 2003

In their native Australia the budgerigar’s breeding season is very short and rapid nest establishment is crucial. To minimize the time required to locate and establish nesting sites, female budgerigars were predicted to use cues on nest site locations provided by other females in their colony. Experimental results from captive birds indicated that, while females can be attracted to nests that are occupied by other females, the social and breeding experience of individuals affects this use of conspecific cues. Females that have had altercations with other females that resulted in physical injuries showed an aversion to apparently occupied nestboxes. Females that had no such social experience prior to testing were attracted to apparently occupied nestboxes. Breeding experience, as well as social experience, may have an effect on the use of conspecifics as cues. Experienced breeding females were less likely to be attracted to apparently occupied nestboxes than were females that had never bred before.