bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity
Authors: Manjunatha U. H. , Mahadevan S. , Visweswariah Sandhya S. , Nagaraja V.
Published: March 2003

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross‐reactivity with both fast‐growing and slow‐growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N‐terminus (residues 351–415) of the enzyme in a conformation‐dependent manner. These monoclonal antibodies would serve as valuable tools for structure–function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.
Acidic pH generated by H+‐ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum
Authors: Monni Marco , Roberti Rita , Corazzi Lanfranco
Published: March 2003

Fusogenic protein (FP) is a glycoprotein (≈ 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti‐FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase‐treated microsomes showed that the proteolysis removed a fragment (≈ 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+‐ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+‐translocating ATPase activity.
Novel activator protein‐2α splice‐variants function as transactivators of the ovine placental lactogen gene
Authors: Limesand Sean W. , Anthony Russell V.
Published: March 2003

Activator protein‐2 (AP‐2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP‐2 cis‐acting element has been described for other genes expressed in the placenta, but the AP‐2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (−124 bp to +16 bp) by AP‐2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP‐2α was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3‐kb transcript was identified by Northern hybridization. Four nearly full‐length AP‐2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP‐2 mRNA expressed in the ovine placenta shares identity with human AP‐2α, but variations in the predicted N‐terminus were observed, and three unique AP‐2α splice‐variants were identified. Expression of AP‐2α variants in HepG2 cells, devoid of endogenous AP‐2, indicates that enhancement through the AP‐2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP‐2α splice‐variants were confirmed in ovine placenta by RT‐PCR, and homologs for two variants were found in human placenta. However, only one AP‐2α transcript, which shares identity to Xenopus AP‐2α, was expressed in BeWo cells. Immunoblot analysis confirmed AP‐2α variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP‐2α variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP‐2α splice‐variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.
GroEL‐assisted refolding of adrenodoxin during chemical cluster insertion
Authors: Iametti Stefania , Bera Aloke K. , Vecchio Giuseppe , Grinberg Asya , Bernhardt Rita , Bonomi Francesco
Published: March 2003

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant‐unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfurtransferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese‐like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH–cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin‐containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.
Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ‐15
Authors: Kim Byung‐Taek , Hong Sung‐Woon , Kim Wan‐Seok , Kim Yeong Shik , Kim Dong‐Hyun
Published: March 2003

Two novel acharan sulfate lyases (ASL1 and ASL2: no EC number) have been purified from Bacteroides stercoris HJ‐15 which was isolated from human intestinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzymes were purified to apparent homogeneity by a combination of QAE‐cellulose, DEAE‐cellulose, carboxymethyl–Sephadex C‐50, hydroxyapatite and HiTrap SP Sephadex C‐25 column chromatography with the final specific activity of 50.5 and 76.7 µmol·min−1·mg−1, respectively. Both acharan sulfate lyases are single subunits of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed optimal activity at pH 7.2 and 45 °C. ASL1 activity was inhibited by Cu2+, Ni2+ and Co2+, but ASL2 activity was inhibited by Cu2+, Ni2+and Pb2. Both enzymes were slightly inhibited by some agents that modify histidine and cysteine residues, but activated by reducing agents such as dl‐dithiothreitol and 2‐mercaptoethanol. Both purified bacteroidal acharan sulfate lyases acted to the greatest extent on acharan sulfate, and to a lesser extents on heparan sulfate and heparin. They did not act on de‐O‐sulfated acharan sulfate. These findings suggest that the biochemical properties of these purified acharan sulfate lyases are different from those of the previously purified heparin lyases, but these enzymes belong to heparinase II.
HMG‐1 enhances HMG‐I/Y binding to an A/T‐rich enhancer element from the pea plastocyanin gene
Authors: Webster Carl I. , Packman Leonard C. , Gray John C.
Published: March 2003

High‐mobility‐group proteins HMG‐1 and HMG‐I/Y bind at overlapping sites within the A/T‐rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG‐1 enhanced the binding of HMG‐I/Y to a 31‐bp region (P31) of the enhancer. Circularization assays showed that HMG‐1, but not HMG‐I/Y, was able to bend a linear 100‐bp DNA containing P31 so that the ends could be ligated. HMG‐1, but not HMG‐I/Y, showed preferential binding to the circular 100‐bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG‐1 was not responsible for enhanced binding of HMG‐I/Y. Direct interaction of HMG‐I/Y and HMG‐1 in the absence of DNA was demonstrated by binding of 35S‐labeled proteins to immobilized histidine‐tagged proteins, and this was due to an interaction of the N‐terminal HMG‐box‐containing region of HMG‐1 and the C‐terminal AT‐hook region of HMG‐I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG‐1 had an affinity for immobilized HMG‐I/Y (Kd = 28 nm) similar to that for immobilized P31 DNA. HMG‐1‐enhanced binding of HMG‐I/Y to the enhancer element appears to be mediated by the formation of an HMG‐1–HMG‐I/Y complex, which binds to DNA with the rapid loss of HMG‐1.
Poly(A) polymerase from Escherichia coli adenylylates the 3′‐hydroxyl residue of nucleosides, nucleoside 5′‐phosphates and nucleoside(5′)oligophospho(5′)nucleosides (NpnN)
Authors: Sillero María A. Günther , Socorro Sónia , Baptista María J. , del Valle Mercedes , de Diego Anabel , Sillero Antonio
Published: March 2003

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3′‐OH residue of a variety of nucleosides, nucleoside 5′‐phosphates and dinucleotides of the type nucleoside(5′)oligophospho(5′)nucleoside is described here for the first time. Using micromolar concentrations of [α‐32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5′)diphospho(5′)adenosine (Ap2A), adenosine (5′)triphospho(5′)adenosine (Ap3A), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), adenosine(5′)pentaphospho(5′)adenosine (Ap5A), guanosine(5′)diphospho(5′) guanosine (Gp2G), guanosine(5′)triphospho(5′)guanosine (Gp3G), guanosine(5′)tetraphospho(5′)guanosine (Gp4G), and guanosine(5′)pentaphospho(5′)guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mm GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.
Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element
Authors: Grad Iwona , Niedziela‐Majka Anita , Kochman Marian , Ożyhar Andrzej
Published: March 2003

The steroid hormone, 20‐hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela‐Majka, A., Kochman, M., Ożyhar, A. (2000) Eur. J. Biochem.267, 507–519] on EcR and Usp DNA‐binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5′ half‐site of the pseudo‐palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition α helix and the roles of the base pairs of the UspDBD target sequence have been probed by site‐directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5′ half‐site of the element may be used for the fine‐tuning of the 20E‐response element specificity and consequently the physiological response.
Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals
Authors: Ricoux Remy , Boucher Jean‐Luc , Mansuy Daniel , Mahy Jean‐Pierre
Published: March 2003

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high‐valent iron‐oxo species which are responsible for both peroxidase‐like and cytochrome P450‐like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2• radicals formed by the oxidation of nitrite by high‐valent iron‐oxo species. This new activity of MP8 opens a new access to nitro‐aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO‐derived species with biomolecules under oxidative stress conditions.
Caenorhabditis elegans expresses a functional phytochelatin synthase
Authors: Clemens Stephan , Schroeder Julian I. , Degenkolb Thomas
Published: March 2003

The formation of phytochelatins, small metal‐binding glutathione‐derived peptides, is one of the well‐studied responses of plants to toxic metal exposure. Phytochelatins have also been detected in some fungi and some marine diatoms. Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis, wheat and Schizosaccharomyces pombe. Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no. 266513. Here we show that C. elegans indeed expresses a gene coding for a functional phytochelatin synthase. CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock‐out strain and confers phytochelatin synthase activity to these cells. Thus, phytochelatins may play a role for metal homeostasis also in certain animals.
Secretory ribonucleases in the primitive ruminant chevrotain (Tragulus javanicus)
Authors: Breukelman Heleen J. , Jekel Peter A. , Dubois Jean‐Yves F. , Mulder Patty P. M. F. A. , Warmels Hayo W. , Beintema Jaap J.
Published: March 2003

Phylogenetic analyses of secretory ribonucleases or RNases 1 have shown that gene duplication events, giving rise to three paralogous genes (pancreatic, seminal and brain RNase), occurred during the evolution of ancestral ruminants. A higher number of paralogous sequences are present in chevrotain (Tragulus javanicus), the earliest diverged taxon within the ruminants. Two pancreatic RNase sequences were identified, one encoding the pancreatic enzyme, the other encoding a pseudogene. The identity of the pancreatic enzyme was confirmed by isolation of the protein and N‐terminal sequence analysis. It is the most acidic pancreatic ribonuclease identified so far. Formation of the mature enzyme requires cleavage by signal peptidase of a peptide bond between two glutamic acid residues. The seminal‐type RNase gene shows features of a pseudogene, like orthologous genes in other ruminants investigated with the exception of the bovine species. The brain‐type RNase gene of chevrotain is expressed in brain tissue. A hybrid gene with a pancreatic‐type N‐terminal and a brain‐type C‐terminal sequence has been identified but nothing is known about its expression. Phylogenetic analysis of RNase 1 sequences of six ruminant, three other artiodactyl and two whale species support previous findings that two gene duplications occurred in a ruminant ancestor. Three distinct groups of pancreatic, seminal‐type and brain‐type RNases have been identified and within each group the chevrotain sequence it the first to diverge. In taxa with duplications of the RNase gene (ruminants and camels) the gene evolved at twice as fast than in taxa in which only one gene could be demonstrated; in ruminants there was an ≈ fourfold increase directly after the duplications and then a slowing in evolutionary rate.
Thermodynamics of the folding of D‐glyceraldehyde‐3‐phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry
Authors: Liang Yi , Li Jian , Chen Jie , Wang Chih‐chen
Published: March 2003

Thermodynamics of the refolding of denatured d‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone–substrate binding and chaperone‐assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy–entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat‐capacity change of the binding, −156 kJ·mol−1·K−1, at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0–25.0 °C. With increasing temperature from 15.0 to 37.0 °C, the PDI‐assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 °C, the spontaneous reactivation, PDI‐assisted reactivation and intrinsic molar enthalpy change during the PDI‐assisted refolding of GAPDH are not detected.
Thermodynamic analysis of the binding of glutathione to glutathione S‐transferase over a range of temperatures
Authors: Ortiz‐Salmerón Emilia , Yassin Zeyad , Clemente‐Jimenez M. Jose , Las Heras‐Vazquez F. Javier , Rodriguez‐Vico Felipe , Barón Carmen , García‐Fuentes Luis
Published: March 2003

The binding properties of a glutathione S‐transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15–30 °C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH–GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26‐kDa glutathione S‐transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15–30 °C. Among thermodynamic parameters, whereas ΔG° remains practically invariant as a function of temperature, ΔH and ΔS° both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 °C, ΔG° is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 °C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy–entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (ΔCp°) of −0.238 ± 0.04 kcal per K per mol of GSH bound.
Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A)
Authors: Yuasa Keizo , Ohgaru Takanori , Asahina Masatoshi , Omori Kenji
Published: March 2003

We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual‐substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino‐acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue‐specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS‐7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3–1.6 and 2.1–3.9 µm, respectively), the Vmax values differed significantly (RNPDE11A4 >> RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 >> HSPDE11A2 > HSPDE11A3 >> HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants.
Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells
Authors: Oetke Cornelia , Hinderlich Stephan , Brossmer Reinhard , Reutter Werner , Pawlita Michael , Keppler Oliver T.
Published: March 2003

Sialic acids are the most abundant terminal carbohydrate moiety on cell surface glycoconjugates in eukaryotic cells and are of functional importance for many biological ligand–receptor interactions. It is a widely accepted view that sialic acids cannot be efficiently taken up from the extracellular space by eukaryotic cells. To test this assumption, we cultivated two recently identified human hematopoetic cell lines which are hyposialylated due to a deficiency in de novo sialic acid biosynthesis in the presence of N‐acetylneuraminic acid (NeuAc), the most frequently found sialic acid. Surprisingly, NeuAc medium supplementation rapidly and potently compensated for the endogenous hyposialylation in a concentration‐dependent manner, resulting in the presentation of cell surface sialoglycans involved in cell adhesion, virus infection and signal transduction. We provide several lines of experimental evidence that all suggest that NeuAc was neither extracellularly incorporated nor degraded to a less complex sugar before uptake. Importantly, NeuAc induced a marked increase in intracellular CMP‐NeuAc levels in both human cell lines and in primary cells regardless of the prior sialylation status of the cells. Studies employing 9‐[3H]NeuAc revealed an uptake consistent with the observed incorporation of unlabeled NeuAc. We propose the existence of an efficient uptake mechanism for NeuAc in eukaryotic cells.
Assembly of gap junction channels
Authors: Ahmad Shoeb , Martin Patricia E. M. , Evans W. Howard
Published: March 2003

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell‐free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin‐binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin‐binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin‐binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Δ215, when transiently expressed in COS‐7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C‐terminal sequence of Cx32 incorporating the calmodulin‐binding site was required for the formation of hetero‐oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS‐7 cells. The results indicate that amino‐acid sequences in the third transmembrane domain and a calmodulin‐binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.
Essential roles of CD14 and lipopolysaccharide‐binding protein for activation of toll‐like receptor (TLR)2 as well as TLR4
Authors: Muta Tatsushi , Takeshige Koichiro
Published: March 2003

Although genetic studies have revealed a critical role for the toll‐like receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We have found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPS‐induced NF‐κB activation in human embryonic kidney 293 cells. The reconstitution systems we established here allow direct biochemical characterization and comparison of activation of each receptor. TLR4 is ≈ 100‐fold more sensitive to LPS than TLR2. In contrast to the response to commercial LPS preparations, TLR2 is unresponsive to repurified LPS or synthetic lipid A, indicating the requirement for an additional molecule(s). On the other hand, a lipid A‐neutralizing reagent, polymyxin B, blocks the ability of the LPS preparation to stimulate both receptors, suggesting that lipid A is also involved in the activation of TLR2. Mutant TLRs harboring a point mutation in the cytoplasmic domain is inactive in transducing the signal upon stimulation, and act as dominant‐negative mutants specifically inhibiting the activation of corresponding type of the receptor but not the other type. Thus, the two receptors are independently activated by distinguishable ligands. Nevertheless, the responses of both TLRs to the LPS preparation are strongly dependent on serum and CD14 and LPS‐binding protein are essential for the activation of both of the two receptors. Supporting its functional significance, both receptors are found to associate with CD14.
Alternate routes to conformational specificity in a Greek key β barrel protein
Authors: Qi Xiao‐Fei , Bagby Stefan , Gombos Zoltan , Ikura Mitsuhiko , Chakrabartty Avijit
Published: March 2003

The N‐terminal domain of protein S, a Greek key calcium‐binding protein from Myxococcus xanthus, forms an atypical molten globule in the calcium‐free state. The structure of this state is characterized by significant conformational fluctuations, which are localized to a subdomain that is not contiguous along the polypeptide chain. The conformational instability of this subdomain appears to arise from repulsive electrostatic interactions of four acidic side chains that are clustered together but are removed from the calcium‐binding sites. This domain can be induced to form a native‐like state through two different routes, calcium binding or reduction of pH. Acid‐induced folding stabilizes the locally unfolded subdomain by selectively removing repulsive interactions without significantly affecting global stability. In contrast, calcium binding appears to increase local stability indirectly by causing global stabilization.
Presence of a genistein‐responsive inhibitory mechanism on interleukin‐1α‐induced NF‐κB activation
Authors: Tago Kenji , Funakoshi Megumi , Mano Hiroyuki , Yanagisawa Ken , Hayakawa Morisada , Kuroiwa Kenji , Iwahana Hiroyuki , Kasahara Tadashi , Tominaga Shin‐ichi
Published: March 2003

Interleukin 1 (IL‐1) is known to activate the signal transduction machinery, including the transcription factor, nuclear factor kappa B (NF‐κB). The activation mechanism of NF‐κB has been studied intensively, while the negative regulatory mechanisms of NF‐κB remain to be clarified. In the present study, we found that genistein, a tyrosine kinase inhibitor, augmented IL‐1α‐dependent NF‐κB activation, suggesting the presence of a tyrosine kinase mediating a suppression signal on NF‐κB. As determined by luciferase reporter gene assay using κB‐responsive element, genistein enhanced IL‐1α‐induced NF‐κB activation. Although genistein failed to increase luciferase activity at 1 and 3 h after IL‐1α stimulation, it induced prolonged activation beginning at 6 h after the initial stimulation. We next examined whether genistein augmented the DNA‐binding activity of NF‐κB, using electrophoretic mobility shift assay. In the case of the control experiment, the binding of NF‐ κB to the κB‐responsive element peaked at 30 min after IL‐1α stimulation, and decreased thereafter. In contrast, treatment with genistein maintained the maximum binding activity for at least 2 h after stimulation. Moreover, genistein enhanced the IL‐1α‐dependent degradation of IκBα. Taken together, our results indicate that genistein augments IκB degradation, resulting in continuous NF‐κB activation. This suggests the possibility that tyrosine kinase negatively regulates NF‐κB.
Plasticity of Alarm‐call Response Development in Belding’s Ground Squirrels (Spermophilus beldingi, Sciuridae)
Authors: Mateo Jill M. , Holmes Warren G.
Published: March 2003

Plasticity in the alarm‐call responses of Belding’s ground squirrels (Spermophilus beldingi) may function to prepare young to respond appropriately to calls according to the predator environment and habitat in which the young develop. To examine the extent to which antipredator responses are sensitive to early rearing environments, we studied the development of behavioural responses to playbacks of alarm calls and non‐alarm calls in free‐living juveniles and captive juveniles housed in large outdoor enclosures. Compared with same‐aged, free‐living juveniles, captive juveniles were more likely to show an observable response to playbacks, exhibited more exaggerated initial responses (e.g. enter a burrow vs. freeze), and remained alert longer following playbacks. The influence of rearing history on antipredator responses was limited to responses to auditory stimuli, as the two groups of juveniles reacted similarly to fast‐moving visual stimuli. The responses of free‐living juveniles appeared to be more discriminating than responses of captive juveniles, particularly following playbacks of calls associated with less immediate threats.

The responses of captive and free‐living mothers were similar, indicating a developmental component to the juvenile response differences observed here. Free‐living juveniles developed a discrimination among alarm and non‐alarm calls sooner than captive young. Response differences were evident within 1 wk of first emergence from natal burrows and persisted at least 4 wk, at around the age of natal dispersal. This suggests that early rearing history has an enduring effect on response repertoires, which may be adaptive if animals continue to inhabit the predator environment in which they developed.
A Technique for the Temporary Application and Augmentation of Pigment Patterns in Fish
Authors: Hoefler Chad D. , Morris Molly R.
Published: March 2003

Secondary sexual characteristics, such as pigment patterns in male fish, have significant roles in female mating preferences. Vertical body‐bar pigmentation, one such characteristic, is found in a variety of Xiphophorus species. Prior investigations have revealed that these bars function as a signal attracting females and deterring rival males in at least two species. Furthermore, it has been shown that X. cortezi females prefer vertical bar symmetry as well as more bars in conspecific males. Because of the shortcomings of prior techniques used for pigment elimination and permanent color marking, a temporary method for augmenting pigment patterns with an artificial dye was developed. This technique was tested using the known preference that female X. cortezi have for more bars in conspecific males. The results corroborate previous findings as female X. cortezi preferred males with more artificial vertical bars in comparison with males with fewer. Therefore, the method of applying temporary, artificial pigment patterns using an antiseptic dye would appear to be an effective way to control and manipulate secondary sexual pigmentation for behavioral investigations in fish.
Social Interaction is Necessary for Discrimination between and Memory for Odours of Close Relatives in Golden Hamsters
Authors: Todrank Josephine , Heth Giora , Johnston Robert E.
Published: March 2003

The role of social experience in recognition of individuals and their odours is not well understood. In a previous study, hamsters discriminated between the odours of their familiar litter mates, but they did not discriminate between the odours of two males or two females from a different litter that were unfamiliar to them. In this paper the role of social experience in discrimination between odours of male litter mates is examined using habituation techniques. Males, tested 1.5–2 mo after separation from their litter mates, discriminated between the flank gland odours of their foster brothers, but they did not discriminate between flank odours from their unfamiliar brothers that had been reared by another mother (Expt. 1). In Expt. 2, adult males did not discriminate between the flank gland odours of two males from a different litter after a week of exposure across a barrier to their sights, sounds, and odours, but subjects did discriminate between the odours of these same males after five brief encounters with them. A month after the encounters, however, subjects no longer discriminated between these odours. In contrast, adult males discriminated between the flank odours of their brothers 9 mo after separation from them (Expt. 3). These results suggest that hamsters must have direct interactions with closely related individuals to discriminate between their odours because the odours of close kin are so similar. Experience with nest mates results in long‐lasting memories for their odours.
Mate Guarding in the Cricket Gryllodes sigillatus: Influence of Multiple Potential Partners
Authors: Bateman Philip W. , MacFadyen Duncan N.
Published: March 2003

There are several hypotheses as to the function of postcopulatory mate guarding. Control over the mate‐guarding period by either sex could potentially influence relative reproductive success. Mate‐guarding behaviour in Gryllodes sigillatus was studied under several conditions: 1. undisturbed pairs; 2. pairs with a single male intruder; 3. pairs exposed acoustically, visually and olfactorily to several other males; 4. pairs exposed freely to several other males; and 5. pairs exposed freely to several other females. The results provide support for the spermatophore retention and rival defence hypotheses. The efficacy of mate guarding was not compromised by the pair being in acoustic, visual and olfactory contact with several other males. Once pairs were exposed to free contact with several other males, the spermatophore retention time by the female declined significantly, indicating that the mate guarder’s efficiency declines under competition from several rivals. In pairs exposed to contact with several females after mating, the mate‐guarding period and spermatophore retention time declined as the mate guarder abandoned the mated female and pursued the other females. Termination of the effective mate‐guarding period by either sex seems to be influenced by the number of other potential partners present.
Space Use in Response to Conspecifics by the Red‐backed Salamander (Plethodon cinereus, Plethodontidae, Caudata)
Authors: Quinn Vanessa S. , Graves Brent M.
Published: March 2003

The presence of conspecifics, as well as the distribution and quality of resources, can influence habitat settlement decisions of animals. We studied the habitat preference and group size of the red‐backed salamander, Plethodon cinereus, from the Upper Peninsula of Michigan. In contrast to previous work with P. cinereus from Virginia, we found groups of P. cinereus within higher quality habitat than solitary individuals and these groups contained both small and large individuals. Under identical conditions in the laboratory, salamanders from Virginia formed a uniform spatial distribution and individuals from northern Michigan displayed an aggregated spatial distribution. Our results suggest that P. cinereus from northern Michigan preferentially settle near conspecifics, while P. cinereus from Virginia avoid them. Our laboratory experiments also suggest that the aggregation behaviour of P. cinereus from northern Michigan may be the result of attraction to conspecifics rather than attraction to habitat features.
Mimicking Natural Nursing Conditions Promotes Early Pup Survival in Domestic Rabbits
Authors: Coureaud Gérard , Schaal Benoist , Coudert Pierre , Hudson Robyn , Rideaud Patricia , Orgeur Pierre
Published: March 2003

In the wild rabbit, Oryctolagus cuniculus, mother–young relationships are based on restricted, once‐per‐day nursing interactions. Correspondingly, pups have evolved an efficient strategy of energy saving. Here we investigate under breeding conditions, whether matching or not, the once‐daily nursing visit by the rabbit females has an effect on pup survival and growth. Two nursing regimen were applied to 89 primiparous (P) and to 78 multiparous (M) does: (a) one that matched the once daily nursing pattern (closed nest‐box during the whole day except for a few minutes devoted to nursing) and (b) one that did not match it (24 h free nest access). In P females, the controlled nest access resulted in lower mortality between birth and weaning (8.1%) as compared to the free nest‐access (18%). This effect was recorded from postnatal d 3–4 onwards. Both treatments induced different death causes (starvation (63%) in controlled‐access regimen, and wounds and nest‐soiling (29%) in free‐access regimen). While both experimental nest‐access regimens differentially affected pup survival in P or M females, they were without influence on pup growth rate in does of either parity. It is concluded that repeated nest visits by the female increase risks of injury to pups, and of out‐of‐time pup activation or sucking, and that, more generally, it plays against the ethophysiologigal strategy of biomass conservation evolved by rabbit newborns. The fact that the nest‐access regimen no longer affected pup survival from the second parity suggests that the behaviour of multiparous does more adequately models the offspring demands.
Defence of Females by Dominant Males of Artibeus jamaicensis (Chiroptera: Phyllostomidae)
Authors: Ortega Jorge , Arita Héctor T.
Published: March 2003

Defence of females by dominant males of the Jamaican fruit‐eating bat Artibeus jamaicensis was observed in two natural colonies over 2 yr. A log‐linear model was used to evaluate the frequency distribution of visits to harems by sex, season and agonistic interaction of dominant males. Harem group size varied from four to 18 females, with one adult male in the small and medium‐sized groups and two males in the large groups (> 14 females). A highly significant interaction was noted between the age and sex of the visitor and the response of the dominant male. Male visitors were attacked more often than female and juvenile visitors. Aggressive defence increased during the reproductive seasons, with dominant males showing more agonistic responses towards male visitors. An increase in the frequency of visits by male visitors was noted in harem groups that ranged in size from four to 12 females, but the frequency of male visits declined in harem groups that contained more than 14 females.
Intra‐ and Inter‐seasonal Variation in the Socio‐Spatial Behavior of Adult Male Collared Lizards, Crotaphytus collaris (Reptilia, Crotaphytidae)
Authors: Baird Troy A. , Sloan Chris L. , Timanus Dusti K.
Published: March 2003

When individuals maintain strong inter‐seasonal philopatry to the same territories, males may be able to re‐establish territory occupancy without intense intra‐sexual aggression, and instead spend more time courting females early in the reproductive season. Furthermore, when some males have prior experience defending the same territories, it may be necessary for young males to exhibit higher levels of aggression because they are establishing a territory for the first time. We tested these hypotheses by examining within‐season (1992 and 1997) temporal variation in the social behavior of adult male collared lizards of known age and prior territorial experience in a population where inter‐season philopatry to territories is high. Contrary to expectations, the frequency of aggression exhibited by males with and without prior territorial experience did not differ. The frequency of intra‐sexual aggression was higher in 1992 than in 1997, perhaps because male competitors were more abundant in 1992. Although there was an interactive effect of year, male display and patrol were low at the beginning of the reproductive season in Apr. and May, reached peaks during midseason in June, and then decreased as reproduction ended in July. The size of territories showed a similar pattern, with males defending larger areas in June. Our data support the philopatry hypothesis in that the establishment of territories occurred without high levels of aggression early in the season, perhaps because territory boundaries have been well defined by high rates of patrol and advertisement during the middle of the previous season. Inter‐sexual interactions were most frequent in June rather than at the beginning of the reproductive season. Adult females are producing their second clutches and yearling females are producing their first clutches in June. The high rate of inter‐sexual encounters in June supports the hypothesis that males allocate more time to courtship when females are receptive because there are more reproductively active females at this time. The temporal pattern of activities in adult Crotaphytus collaris appears to function as a compromise between competing intra‐ and inter‐sexual social demands on males, allowing males to maximize mating opportunities as well as maintain future access to productive territories.
Anti‐Predator Behavior of Vancouver Island Marmots: Using Congeners to Evaluate Abilities of a Critically Endangered Mammal
Authors: Blumstein Daniel T. , Daniel Janice C. , Bryant Andrew A.
Published: March 2003

Behavioral comparisons between endangered species and their congeners may provide valuable data with which to test ideas about declining populations or the future direction of recovery efforts. We considered the case of the highly endangered Vancouver Island marmot (Marmota vancouverensis). Predation is a current source of mortality, and inadequate anti‐predator behavior could have profound ramifications for the future success of re‐introductions. We tested whetherM. vancouverensisanti‐predator behavior was unusual or ‘deficient’ by quantifying it and comparing it to 13 other marmot species. We found no evidence that Vancouver Island marmots were unwary. If anything, the converse was true. Vancouver Island marmots were responsive and vigilant towards real and simulated predatory threats. They dug numerous escape burrows that reduced the likelihood of predation. Our results have several implications for future recovery efforts, one of which was to establish ‘baseline’ flight‐response targets that captive‐bred Vancouver Island marmots will have to meet or exceed prior to release into predator‐rich environments.
Anti‐Predator Strategies and Grouping Patterns in White‐Tailed Deer and Mule Deer
Authors: Lingle Susan
Published: March 2003

White‐tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) are closely related species of similar size that differ in their anti‐predator behavior. White‐tails flee from coyotes (Canis latrans), whereas mule deer typically stand their ground and attack this predator. I used observations of coyotes hunting deer to identify: (i) changes in group structure made in response to coyotes; and (ii) the relationship between group structure and the risk of predation for each species.

In response to coyotes, groups of mule deer merged with other groups and individuals bunched together. Predation attempts were more likely to escalate when groups split and individuals failed to bunch. Coyotes typically attacked mule deer that were in outlying positions, and these deer had to move to central positions to end attacks. Due to the high frequency of attacks on small groups as well as to the level of dilution of risk, individuals in small mule deer groups were at high risk of being attacked compared with those in larger groups. In contrast to mule deer, white‐tails made no consistent changes in group size or formation, and coyotes attacked individuals in central as well as in outlying positions. Variation in aspects of group cohesion was not related to the vulnerability of white‐tails, and there was no obvious difference in the risk of attack facing individuals in groups of different size. These results suggest that coyote predation selects for relatively large, cohesive groups in mule deer, apparently because this type of group improves their ability to deter coyotes. Coyote predation does not have similar effects on groups formed by white‐tails, which use flight rather than deterrence to avoid predation. The benefits of responding cohesively, occupying certain positions within groups, and forming groups of a certain size can vary widely depending on the anti‐predator strategies used by an animal.
Why is Female Choice not Unanimous? Insights from Costly Mate Sampling in Marine Iguanas
Authors: Wikelski Martin , Carbone Chris , Bednekoff Peter A. , Choudhury Sharmila , Tebbich Sabine
Published: March 2003

Females do not unanimously choose the single ‘best’ male, even when female choice is strong, such as in leks, or in polygynous mating situations. A possible explanation is that females base their choices on limited information, perhaps because gathering information is costly. We tested this hypothesis by continuously observing individual female marine iguanas throughout the mating period in order to document the information they gathered about each potential mate. Females actively visited approximately five additional males during the 3 d prior to copulation, compared to the males seen on their normal foraging routes. Females were more likely to visit large‐bodied males, but preferentially copulated with the male that had the highest display rate of all males they visited. Females that mated on a dense territory cluster mated with more active males than did those that mated on dispersed territories. However, females on a dense cluster also lost more body mass, potentially as a consequence of high rates of interaction with males. This mass loss may represent an important cost and result from postural changes in response to male attention. Such costs may explain why females only gather a certain amount of information and why females on dispersed territories choose less active mates. Lack of complete information introduces subjectivity into female choice: what is perceived as best by one female may not be perceived as best by another. Thus, lack of complete information may prevent unanimity of female choice.