bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis
Authors: Simard Dave , Hewitt Kerry A. , Lunn Faylene , Iyengar Akshai , Bearne Stephen L.
Published: May 2003

Cytidine 5′‐triphosphate synthase catalyses the ATP‐dependent formation of CTP from UTP using either ammonia or l‐glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin‐catalysed proteolysis, Edman degradation, and site‐directed mutagenesis were used to identify peptide bonds C‐terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP‐binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin‐catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin‐catalyzed proteolysis in the GAT domain and wild‐type ability to catalyse NH3‐dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine‐dependent CTP formation were reduced ≈ 20‐fold and ≈ 10‐fold, respectively, relative to wild‐type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6‐fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10‐fold. This suggests that Arg429 plays a role in both GTP‐dependent activation and GTP binding.
Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina
Authors: Maróti Gergely , Fodor Barna D. , Rákhely Gábor , Kovács Ákos T. , Arvani Solmaz , Kovács Kornél L.
Published: May 2003

There are at least two membrane‐bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon‐based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase‐specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane‐bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In‐frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.
The cellular oxygen tension regulates expression of the endoplasmic oxidoreductase ERO1‐Lα
Authors: Gess Bernhard , Hofbauer Karl‐Heinz , Wenger Roland H. , Lohaus Christiane , Meyer Helmut E. , Kurtz Armin
Published: May 2003

The formation of disulfide bonds in the endoplasmic reticulum requires protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductin 1 (ERO1) that reoxidizes PDI. We report here that the expression of the rat, mouse and human homologues of ERO1‐Like protein α but not of the isoform ERO1‐Lβ are stimulated by hypoxia in rats vivo and in rat, mouse and human cell cultures. The temporal pattern of hypoxic ERO1‐Lα induction is very similar to that of genes triggered by the hypoxia inducible transcription factor (HIF‐1) and is characteristically mimicked by cobalt and by deferoxamine, but is absent in cells with a defective aryl hydrocarbon receptor translocator (ARNT, HIF‐1β). We speculate from these findings that the expression of ERO1‐Lα is probably regulated via the HIF‐pathway and thus belongs to the family of classic oxygen regulated genes. Activation of the unfolded protein response (UPR) by tunicamycin, on the other hand, strongly induced ERO1‐Lβ and more moderately ERO1‐Lα expression. The expression of the two ERO1‐L isoforms therefore appears to be differently regulated, in the way that ERO1‐Lα expression is mainly controlled by the cellular oxygen tension, whilst ERO1‐Lβ is triggered mainly by UPR. The physiological meaning of the oxygen regulation of ERO1‐Lα expression likely is to maintain the transfer rate of oxidizing equivalents to PDI in situations of an altered cellular redox state induced by changes of the cellular oxygen tension.
Co‐operation of domain‐binding and calcium‐binding sites in the activation of gelsolin
Authors: Lagarrigue Emeline , Maciver Sutherland K. , Fattoum Abdellatif , Benyamin Yves , Roustan Claude
Published: May 2003

Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a ‘helical‐latch’ at the C‐terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium‐free compact state, and that the crutial calcium binding site(s) reside in the C‐terminal half of gelsolin perhaps involving the C‐terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (Kd = ≈15 µm). We also report a calcium dependent binding site for the C‐terminus (G4–6) within G2 and delimit this further to a specific region formed by residues 203–225 and 159–193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium‐dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4–6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.
The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13
Authors: Divari Sara , Valetti Francesca , Caposio Patrizia , Pessione Enrica , Cavaletto Maria , Griva Ersilia , Gribaudo Giorgio , Gilardi Gianfranco , Giunta Carlo
Published: May 2003

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono‐hydroxylation of substrates (phenol, and with less efficiency, chloro‐ and methyl‐phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: α (54 kDa), β (38 kDa) and γ (11 kDa). The gene encoding PHO α (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO α contains the enzyme active site, harbouring a dinuclear iron centre Fe‐O‐Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the α‐subunit. The prevalence of α‐helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 °C and finally the pH for optimal activity was determined to be 7.5.
The histidine‐phosphocarrier protein ofStreptomyces coelicolorfolds by a partially folded species at low pH
Authors: Fernández‐Ballester Gregorio , Maya Javier , Martín Alejandro , Parche Stephan , Gómez Javier , Titgemeyer Fritz , Neira José L.
Published: May 2003

The folding of a 93‐residue protein, the histidine‐phosphocarrier protein ofStreptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8‐anilinonaphthalene‐1‐sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical‐denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two‐state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ΔG = 4.0 ± 0.2 kcal·mol−1(1 kcal = 4.18 kJ), which makes the HPr fromS. coelicolorthe most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7–9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5–4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native‐like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8‐anilinonaphthalene‐1‐sulfate binding experiments); or (b) that measured by acquisition of native‐like compactness (gel‐filtration studies). It seems that acquisition of native‐like features occurs in a wide pH range and it cannot be ascribed to a unique side‐chain titration. These series of intermediates have not been reported previously in any member of the HPr family.
Gene transcription of fgl2 in endothelial cells is controlled by Ets‐1 and Oct‐1 and requires the presence of both Sp1 and Sp3
Authors: Liu Mingfeng , Leibowitz Julian L. , Clark David A. , Mendicino Michael , Ning Qin , Ding Jin Wen , D'Abreo Cheryl , Fung Laisum , Marsden Philip A. , Levy Gary A.
Published: May 2003

The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome. Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages. To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5′‐RACE and primer extension. A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription. A complex positive regulatory domain (PRD) spanning a 39‐bp sequence from −87 to −49 (relative to the transcription start site) was identified. Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct‐1, and Ets‐1. Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets‐1 and requires the presence both of the Sp1 and Sp3 transcription factors. The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated. Moreover, viral induced fgl2 expression also requires the presence of this PRD. These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral‐induced fgl2 expression in endothelial cells.
A peptide containing a novel FPGN CD40‐binding sequence enhances adenoviral infection of murine and human dendritic cells
Authors: Richards Julie L. , Abend Johanna R. , Miller Michelle L. , Chakraborty‐Sett Shikha , Dewhurst Stephen , Whetter Linda E.
Published: May 2003

CD40 is a receptor with numerous functions in the activation of antigen presenting cells (APCs), particularly dendritic cells (DC). Using phage display technology, we identified linear peptides containing a novel FPGN/S consensus sequence that enhances the binding of phage to a purified murine CD40‐immunoglobulin (Ig) fusion protein (CD40‐Ig), but not to Ig alone. To examine the ability the FPGN/S peptides to enhance adenoviral infection of CD40‐positive cells, we used bifunctional peptides consisting of an FPGN‐containing peptide covalently linked to an adenoviral knob‐binding peptide (KBP). One of these, FPGN2‐KBP, was able to enhance adenoviral infection of both murine and human DCs in a dose‐dependent manner. FPGN2‐KBP also improved infection of murine B cell blasts, a murine B lymphoma cell line (L10A), and immortalized human B cells. To demonstrate that enhancement of adenoviral infection depended on the presence of CD40, we analyzed infection of the breast cancer line, SKBR3, that does not express CD40 or the adenovirus cellular receptor, CAR. Infection of SKBR3 cells was enhanced by FPGN2‐KBP following transient transfection with a plasmid vector that expresses murine CD40, but not when the cells were mock‐transfected. In conclusion, we have isolated a peptide that binds to murine CD40, and promotes the uptake of adenoviruses into CD40‐expressing cells of both murine and human origin, suggesting that it may have potential applications for antigen delivery to CD40‐positive antigen‐presenting cells.
Cytochrome b559 content in isolated photosystem II reaction center preparations
Authors: Yruela Inmaculada , Miota Francisca , Torrado Elena , Seibert Michael , Picorel Rafael
Published: May 2003

The cytochrome b559 content was examined in five types of isolated photosystem II D1‐D2‐cytochrome b559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized‐minus‐reduced difference extinction coefficient of cytochrome b559 at 559 nm. The relative amount of D1 and cytochrome b559α‐subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b559α‐subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII‐enriched membrane fragments used as the RC‐isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their α‐subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1‐control sample calibration curve gave a ratio of 1.25 cytochrome b559α‐subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes.
Changes in rat liver mitochondria with aging
Authors: Bakala Hilaire , Delaval Evelyne , Hamelin Maud , Bismuth Jeanne , Borot‐Laloi Caroline , Corman Bruno , Friguet Bertrand
Published: May 2003

Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was measured and the accumulation of oxidation and glycoxidation (Nε‐carboxymethyllysine, CML) products was monitored using immunochemical assays. The function of isolated mitochondria was assessed by measuring respiratory chain activity. Mitochondria from aged (27 months) rats exhibited the same rate of oxygen consumption as those from adult (10 months) rats without any change in coupling efficiency. At the same time, the ATP‐stimulated Lon protease activity, measured as fluorescent peptides released, markedly decreased from 10‐month‐old rats (1.15 ± 0.15 FU·µg protein−1·h−1) to 27‐month‐old‐rats (0.59 ± 0.08 FU·µg protein−1·h−1). In parallel with this decrease in activity, oxidized proteins accumulated in the matrix upon aging while the CML‐modified protein content assessed by ELISA significantly increased by 52% from 10 months (11.71 ± 0.61 pmol CML·µg protein−1) to 27 months (17.81 ± 1.83 pmol CML·µg protein−1). These results indicate that the accumulation of deleterious oxidized and carboxymethylated proteins in the matrix concomitant with loss of the Lon protease activity may affect the ability of aging mitochondria to respond to additional stress.
Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells
Authors: Frick Inga‐Maria , Schmidtchen Artur , Sjöbring Ulf
Published: May 2003

Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface‐expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2‐terminal part as well as in the C‐repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC‐chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein‐mediated adhesion of S. pyogenes.
Crystal structure of thiamindiphosphate‐dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole‐3‐acetic acid
Authors: Schütz Anja , Sandalova Tatyana , Ricagno Stefano , Hübner Gerhard , König Stephan , Schneider Gunter
Published: May 2003

The thiamin diphosphate‐dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole‐3‐acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 Å resolution and refined to a crystallographic R‐factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open α/β topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20° in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.
Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway
Authors: Schütz Anja , Golbik Ralph , Tittmann Kai , Svergun Dmitri I. , Koch Michel H. J. , Hübner Gerhard , König Stephan
Published: May 2003

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole‐3‐acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole‐3‐acetic acid, catalyses the formation of indole‐3‐acetaldehyde and carbon dioxide from indole‐3‐pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole‐3‐pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped‐flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole‐3‐pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X‐ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH‐dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207–1216] and comparison with indole‐3‐pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.
Covalent and three‐dimensional structure of the cyclodextrinase from Flavobacterium sp. no. 92
Authors: Fritzsche Hanna B. , Schwede Torsten , Schulz Georg E.
Published: May 2003

Starting with oligopeptide sequences and using PCR, the gene of the cyclodextrinase from Flavobacterium sp. no. 92 was derived from the genomic DNA. The gene was sequenced and expressed in Escherichia coli; the gene product was purified and crystallized. An X‐ray diffraction analysis using seleno‐methionines with multiwavelength anomalous diffraction techniques yielded the refined 3D structure at 2.1 Å resolution. The enzyme hydrolyzes α(1,4)‐glycosidic bonds of cyclodextrins and linear malto‐oligosaccharides. It belongs to the glycosylhydrolase family no. 13 and has a chain fold similar to that of α‐amylases, cyclodextrin glycosyltransferases, and other cyclodextrinases. In contrast with most family members but in agreement with other cyclodextrinases, the enzyme contains an additional characteristic N‐terminal domain of about 100 residues. This domain participates in the formation of a putative D2‐symmetric tetramer but not in cyclodextrin binding at the active center as observed with the other cyclodextrinases. Moreover, the domain is located at a position quite different from that of the other cyclodextrinases. Whether oligomerization facilitates the cyclodextrin deformation required for hydrolysis is discussed.
Female Baboons’ Responses to Male Loud Calls
Authors: Kitchen Dawn M. , Cheney Dorothy L. , Seyfarth Robert M.
Published: May 2003

Male baboons (Papio cynocephalus ursinus) give loud, two‐syllable ‘wahoo’ calls in response to predators (alarm wahoos) and during aggressive displays that may include multiple males chasing each other or females (contest wahoos). Although acoustic analysis has revealed significant differences between the two calls, these differences are subtle and the two subtypes can be difficult for humans to distinguish. Whatever the evolutionary mechanisms that might have acted on the production of acoustically graded loud calls, it would seem to be adaptive for listeners to discriminate among calls that are given in qualitatively different contexts. This is particularly true in the case of female baboons. Alarm wahoos, which are given during predator encounters, demand qualitatively different responses from contest wahoos, which are given in contexts when females are at risk of harassment and infanticide by males. In playback experiments, females responded for significantly longer durations to alarm than to contest wahoos. Moreover, only alarm wahoos caused females to flee. Despite their acoustic similarity, female baboons appear to associate alarm and contest wahoos with qualitatively different events.
Testing an Adaptive Hypothesis Through Context‐Dependence: Effects of Water Depth on Foraging Behaviour in Three Species of Garter Snakes
Authors: de Queiroz Alan
Published: May 2003

Adaptive hypotheses based on interspecific comparisons can be tested by evaluating the context‐dependence of the behaviour of individual organisms. Drummond (Behaviour, 86, 1983, 1) categorized garter snake species (Thamnophis) as terrestrial–aquatic generalists or aquatic specialists based on diet and aquatic foraging behaviour. He hypothesized that the characteristic foraging behaviours of aquatic specialists – including frequent crawling on the underwater substrate and a high rate of underwater predatory strikes – are adaptations for feeding on relatively widely dispersed aquatic prey. Drummond's hypothesis based on interspecific comparisons suggests that individual snakes might change their foraging in the direction of aquatic specialist behaviour with an increase in water depth (which increases prey dispersion). I tested this prediction through laboratory observations of Mexican Pacific lowlands garter snakes (T. validus) feeding on minnows in shallow (2 cm) and deep (3–7 cm) water. Members of this species are appropriate subjects because they are ecologically intermediate between the generalists and aquatic specialists studied by Drummond, and thus might be expected to show more variation in aquatic foraging behaviour than those species. T. validus showed significantly higher frequencies of crawling on the underwater substrate and of underwater strikes in the deep water than in the shallow water; i.e. increased water depth shifted the behaviour of these snakes toward that of aquatic specialists, thus supporting Drummond's hypothesis. Individuals of an aquatic specialist species, the narrow‐headed garter snake (T. rufipunctatus), showed less pronounced changes in behaviour with increased water depth. Western ribbon snakes (T. proximus), which feed primarily at the land–water interface (and are expected to act like terrestrial–aquatic generalists), typically refused to feed in deep water. Interspecific differences in underwater visual acuity may underly the behavioural differences among the three species by determining whether changes in foraging behaviour with water depth are advantageous. Information on phylogenetic relationships suggests that the facultative behaviour of T. validus may represent an intermediate stage in the evolution of aquatic specialization.
Male Competition over Access to Females in a Spider with Last‐Male Sperm Precedence
Authors: Schaefer Daniela , Uhl Gabriele
Published: May 2003

Agonistic behaviour between male cellar spiders (Pholcus phalangioides) was investigated to test whether (1) size difference determines which male achieves access to the female, (2) males are able to monopolize access to the female until egg laying and whether (3) female resource value increases before egg laying because of last‐male sperm precedence. We further investigated whether (4) there is variation in time and energy spent on courtship and copulation depending on the degree of sperm competition, i.e. with or without rival present. In three experimental settings we introduced two males of either different or similar sizes, or a single male to a female. The mating units were constantly video‐observed until the females produced their first egg sac. Experience, ownership and female resource value in terms of body size was controlled. Our results show that larger males achieve almost exclusive access to females. Size symmetrical settings resulted in increased fighting activity and duration but dominance did not influence mating success. If copulations were disturbed by the rival male, copulations were terminated earlier in symmetrical settings compared with asymmetrical settings. In 94.8% of trials only one copulation took place, suggesting that the copulating male successfully monopolized access to the female. Males confronted with a rival copulated longer but courted significantly shorter than lone males. Although the last male to copulate sires 88% of the offspring in P. phalangioides, neither fighting nor courtship activity increased before the female laid a batch of eggs. This suggests that males have no indication of the timing of oviposition.
Bat Avoidance in Non‐Aerial Insects: The Silence Response of Signaling Males in an Acoustic Moth
Authors: Greenfield Michael D. , Baker Michelle
Published: May 2003

While the evasive responses of many flying acoustic insects to aerial‐hawking bats are duly recognized and studied, the responses of non‐aerial insects to gleaning bats are generally overlooked. It has been assumed that acoustic insects are deaf to these predators because gleaning bat echolocation calls are typically low in amplitude, brief (1–3 ms) and very high in frequency (>60 kHz). We tested this assumption in a series of playback experiments with a moth (Achroia grisella) that uses hearing in both predator evasion and mating. We report that ultrasound pulses ≥78 dB peSPL (peak equivalent sound pressure level) and ≥1 ms in duration inhibit stationary males from broadcasting their own ultrasonic advertisement calls, provided that the pulsed stimuli are delivered at a repetition rate ≤30/s. Further analyses suggest that inhibition by pulsed ultrasound comprises two processes performed serially. First, a startle response with a latency <50 ms is elicited by a single pulse ≥1 ms duration. Here, a male misses broadcasting several calls over a 50–100 ms interval. Secondly, the startle may be extended as a silence response lasting several to many seconds if subsequent pulses occur at a rate ≤30/s. Call inhibition cannot represent a simple response to acoustic power because of the inverse interaction between pulse duration and rate. On the other hand, the temporal and energy characteristics of inhibitory stimuli match those of gleaning bat echolocation calls, and we infer that inhibition is a specialized defensive behavior by which calling males may avoid detection by eavesdropping bats.
Effect of Group Size on Individual and Collective Vigilance in Greater Rheas
Authors: Fernández Gustavo J. , Capurro Angel F. , Reboreda Juan C.
Published: May 2003

We studied the effect of group size on the proportion of time that greater rheas, Rhea americana, allocated to vigilance and feeding during the non‐breeding season. We tested whether: (1) the proportion of time that one bird allocates to vigilance (individual vigilance) decreases with group size, and (2) the proportion of time that at least one bird of the group is vigilant (collective vigilance) increases with group size. We analyzed video‐recordings of birds that were foraging alone or in groups from two to 12 birds. The proportion of time allocated to individual vigilance decreased and the proportion of time spent feeding increased with group size. In both cases the main significant difference was between birds foraging alone and in groups. Collective vigilance did not vary with group size and it was lower than expected if vigilance bouts were random or sequential. Our results indicate that rheas foraging in large groups would not receive the benefit of an increase in collective vigilance, although they could still benefit from a reduction of predation risk by the dilution effect.
Maternal Separation in Guinea‐Pigs: A Study in Behavioural Endocrinology
Authors: Wewers Dirk , Kaiser Sylvia , Sachser Norbert
Published: May 2003

The aim of this study was to elucidate the modulation of behaviour and endocrine stress responses of guinea‐pig pups by social and spatial factors in a maternal separation paradigm. The animals were kept in two colonies (each colony: nine males, 13 females and their offspring; enclosure size: 6 m2). Blood samples were taken from the ear vessels of eight male and eight female pups (aged 13–14 d) immediately before and 2 h after they were removed from their colony and were placed singly into a novel environment. Furthermore, eight male and eight female pups were tested in their home colony before and after 2 h of sepration from their mothers. Blood samples were taken from control animals (eight male and eight female pups) which remained in the home colonies together with their mothers. Additionally, the behaviour of 16 male and 16 female pups was recorded in their home colonies when their mothers were either present or absent. Male and female pups separated from their mothers showed a significantly higher locomotor activity, with females showing higher frequencies of distress calls, than pups whose mothers were present. The mother's absence did not cause a significant increase in serum cortisol concentrations in the male or the female offspring. This result is entirely different from the endocrine stress response of pups that were placed singly in an unknown environment. These animals showed a significant increase in their serum cortisol concentrations after 2 h of separation from their mothers. The absence of the mother led to distinct changes in the behaviour of the guinea‐pig pups. However, staying in the familiar social and spatial environment buffered the endocrine stress response that normally occurs because of maternal separation.
State of fallen logs and the occurrence of conifer seedlings and saplings in boreal and subalpine old‐growth forests in Japan
Authors: Narukawa Yoshiko , Iida Shigeo , Tanouchi Hiroyuki , Abe Shin , Yamamoto Shin‐Ichi
Published: May 2003

Fallen logs on the forest floor play an important role in tree seedling establishment in boreal and subalpine old‐growth forests. We examined the abundance and state (degree of decay, moss coverage on their surfaces) of fallen logs on the forest floor and compared the occurrence of conifer seedlings and saplings on soil and fallen logs in old‐growth evergreen boreal (Taisetsu) and subalpine (Yatsugatake) coniferous forest stands in Japan. The forest floors of both stands were covered mostly by soil, and fallen logs covered only a small proportion (4.5% in Taisetsu and 2.5% in Yatsugatake) of the forest floor. In both stands, no seedlings were found on freshly fallen logs. Both moss coverage and the occurrence of seedlings or saplings on fallen logs increased with decay in both stands. In Taisetsu, the occurrence of all conifer seedlings and saplings was, in general, restricted to fallen logs. In contrast, in Yatsugatake, Picea and Tsuga seedlings occurred much more frequently on fallen logs than on soil, whereas Abies seedlings and saplings became established on both soil and fallen logs. Seedlings were taller on decayed fallen logs, but taller saplings were rare on fallen logs in Yatsugatake. Results indicate that species’ characteristics, the condition of the fallen logs and environmental factors determine the preponderance of seedling and sapling establishment on fallen logs. The condition of fallen logs varies with the degree of decay, and fallen logs that function as seedling establishment sites on the forest floor are thought to be limited by time and space.
Importance of topography and soil texture in the spatial distribution of two sympatric dipterocarp trees in a Bornean rainforest
Authors: Itoh Akira , Yamakura Takuo , Ohkubo Tatsuhiro , Kanzaki Mamoru , Palmiotto Peter A. , LaFrankie James V. , Ashton Peter S. , Lee Hua Seng
Published: May 2003

Relationships between spatial distributions and site conditions, namely topography and soil texture, were analyzed for two congeneric emergent trees, Dryobalanops aromatica and Dryobalanops lanceolata (Dipterocarpaceae), in a tropical rainforest in Sarawak, East Malaysia. A 52‐ha permanent plot was divided into 1300 quadrats measuring 20 m × 20 m; for each Dryobalanops species, the number and total basal area of trees ≥1 cm in d.b.h. were compared among groups of quadrats with different site conditions. Because spatial distributions of both Dryobalanops and site‐condition variables were aggregated, Monte‐Carlo permutation tests were applied to analyze the relationships. Both single and multifactor statistical tests showed that the density and basal area distributions of the two species were significantly non‐random in relation to soil texture and topographic variables. D. aromatica was significantly more abundant at higher elevations, in sandy soils, and on convex and steep slopes. In contrast, D. lanceolata preferred lower elevations and less sandy soils. In the study plot, there were very few sites (3 of 1150 quadrats tested) where the models of Hayashi's method predicted the co‐occurrence of the two species. These results suggest that between‐species differences in habitat preferences are so large that they alone explain the spatially segregated distributions of these two species within the 52‐ha study plot.
Fine root biomass, production and turnover in a fertilized Larix leptolepis plantation in central Korea
Authors: Son Yowhan , Hwang Jae Hong
Published: May 2003

The effects of fertilization [control (C), 200 kg N ha−1 + 25 kg P ha−1 (LNP) and 400 kg N ha−1 + 50 kg P ha−1 (HNP)] on fine root dynamics were examined in a 40‐year‐old Larix leptolepis plantation in central Korea. The average fine root biomass during the growing season for C, LNP and HNP was 957, 934 and 814 kg ha−1, respectively, whereas the fine root production for C, LNP and HNP was 2103, 2131 and 2066 kg ha−1, respectively. Nitrogen and P inputs into the soil via fine root turnover for C, LNP and HNP were 23.0 and 1.2, 23.3 and 1.2 and 22.6 and 1.2 kg ha−1, respectively. There were no significant differences in fine root biomass, production and N and P inputs through fine root turnover between the fertilization treatments during the first growing season after fertilization.
Safety from thromboembolism using intravenous immunoglobulin therapy in Kawasaki disease: Study of whole‐blood viscosity
Authors: Nishikawa Miki , Ichiyama Takashi , Hasegawa Masanari , Kawasaki Kozo , Matsubara Tomoyo , Furukawa Susumu
Published: May 2003

Abstract
Background
: High‐dose intravenous infusions of immunoglobulin (IVIG) are well established as a standard therapy for Kawasaki disease (KD) for reducing the risk of coronary artery aneurysms. IVIG therapy might increase the blood viscosity both in vitro and in vivo, which has been reported as a risk factor for cardiovascular or cerebrovascular thromboembolism in adults.

Methods
: We measured the whole‐blood viscosity in vitro, serum IgG and albumin, and blood hematocrit in 10 patients with KD and 10 with non‐KD (five with acute encephalitis, one with sepsis, one with idiopathic thrombocytopenic purpura, one with Guillain−Barré syndrome, one with insulin‐dependent diabetes mellitus, and one with Evans syndrome) before and after IVIG therapy.

Results
: The blood viscosity increased significantly after IVIG therapy in the patients with non‐KD, but did not increase in those with KD.

Conclusion
: The results of the present study suggest that the use of IVIG therapy for KD might be relatively safe, with no risk of thromboembolism due to hyperviscosity.
Tension‐type headache in children: A clinical evaluation
Authors: Sarioglu Berrak , Erhan Elvan , Serdaroglu Gul , Doering Birgul Gokce , Erermis Serpil , Tutuncuoglu Sarenur
Published: May 2003

Abstract
Background
: Anxiety and psychological stress are often pre‐existing factors which can trigger pediatric tension‐type headaches.

Methods
: Sixteen children (10 girls and six boys) aged between 7 and 18 years who fulfilled the International Headache Society (IHS) criteria for episodic tension‐type headache were included in the present study. Pain characteristics, associated symptoms, and stress‐triggering factors were evaluated. Psychiatric and psychosocial evaluations were performed according to DSM‐IV criteria.

Results
: Pain was bilateral in 93.7% of patients and bitemporal in 50% of children. The intensity of pain increased with motion and stress in more than half of the patients, while pain decreased with rest and massage in 43.7% of patients. Ten of the 16 (62.5%) patients were diagnosed as having a psychiatric disorder. The most common stress‐triggering factors were difficulty in adaptation at school and relationship problems with family members. All of the children reported 26 stress factors. Of these stress factors, 20 (76.9%) were reported by children diagnosed with psychiatric disorder.

Conclusion
: These results suggest that in children with tension‐type headache a thorough psychiatric evaluation should be performed to rule out underlying psychiatric disorders.
Pharmacokinetics and dosing of arbekacin in preterm and term newborn infants
Authors: Suzuki Keiji , Tanikawa Kouji , Matsuzaki Takashi
Published: May 2003

Abstract
Background
: The objective of the present study was to determine pharmacokinetic variables and to characterize a new initial dosing regimen of arbekacin (ABK) for preterm and term newborn infants.

Patients and methods
: Subjects were 40 infants treated with ABK in a tertiary care neonatal unit over a period of 18 months. At birth, the infants were 23 5/7−40 0/7 weeks and weighed 530−3428 g. Serum ABK concentration was measured at two points in a course of treatment. Data were analyzed by a one‐compartment model to obtain volume of distribution (Vd) and clearance (CL) of ABK. These variables were correlated with the patients’ demographic and laboratory data. The new initial dosing regimen was determined based on these data.

Results
: Sixty pairs of blood samples were taken from the infants. They were divided into three groups: preterm early (PE), gestational age (GA) < 37 weeks and postnatal age (PNA) < 28 days; preterm late (PL), GA < 37 weeks and PNA ≥ 28 days; and term (T), GA ≥ 37 weeks and PNA < 28 days. The Vd was 0.50 ± 0.02, 0.48 ± 0.04, and 0.43 ± 0.03 L/kg, and CL was 0.59 ± 0.04, 1.12 ± 0.10, and 0.78 ± 0.09 mL/min per kg (mean ± SEM) in PE, PL, and T, respectively. The new dosing regimen is 5 mg/kg every 48 h, 5 mg/kg every 24 h, and 4 mg/kg every 24 h for PE, PL, and T, respectively.

Conclusions
: With the new dosing regimen, more infants achieved serum ABK levels within the optimal range than the conventional one.
Use of intramuscular ketamine for endoscopy sedation in children
Authors: Law Albert K , Ng Daniel K , Chan Keung‐kit
Published: May 2003

Abstract
Objective
: To report the experience of the use of intramuscular (IM) ketamine for endoscopy sedation in children.

Methodology
: Children over 6 months of age scheduled for elective endoscopy − esophagogastroduodenoscopy (EGD), bronchoscopy and nasopharyngolaryngoscopy (NPL) were enrolled for (2 mg/kg to 3 mg/kg) intramuscular ketamine sedation. A repeated dose of 2 mg/kg intramuscular ketamine was administered to those who failed the first sedation. Alternative sedation (intravenous midazolam and fentanyl) was given to children who failed ketamine sedation twice. Sedation was regarded as successful if the procedure was completed by endoscopist with a single dose of ketamine.

Results
: Sixty children were enrolled for the study. Overall success rate in our patients was 78.3%. Failure rate in infants was 50%, i.e. 4 out of 8. For children aged 1−7, the failure rate was 32%. Failure rate dropped markedly to 6.7% for those older than 7 years of age, and it showed significant difference when compared with the other two groups. Two cases of laryngospasm were experienced in the present study.

Conclusion
: Intramuscular ketamine is an effective medication for sedation in endoscopy undertaken in children over age 7 years, but it should be avoided with children under age 7 because of the high failure rate.
Mass screening for early detection of congenital kidney and urinary tract abnormalities in infancy
Authors: Yoshida Junko , Tsuchiya Masami , Tatsuma Noriko , Murakami Mutsumi
Published: May 2003

Abstract
Background:
Recent widespread use of ultrasound has led to new efforts at screening for congenital kidney and urinary tract abnormalities. However, a standard screening methodology, criteria defining abnormalities, and follow‐up procedures remain to be established. In order to establish screening criteria for these abnor­malities, we performed a preliminary study in 800 1‐month‐old infants using provisional methods and criteria.

Methods:
Based on the results of preliminary study, we screened 2700 1‐month‐old infants in a prospective study using the criteria of renal size (longitudinal diameter ≤35 mm or ≥60 mm, or a difference between sides of ≥10 mm), and of pelvic dilatation (Society for Fetal Urology [SFU] grade 2 or higher) as positive at the first ultrasound screening. We used the SFU grading system instead of anteroposterior pelvic diameter measurements for pelvic dilatation.

Results:
One hundred and twelve (4.1%) of the 2700 infants had abnormalities at the first ultrasound screening, while 18 (0.67%) had congenital kidney and urinary tract abnormalities on further examination. Use of the SFU grading system enabled us to reduce the false‐positive rate at first screening, while maintaining a high diagnostic rate. The abnormalities consisted of ureteropelvic junction obstruction in seven infants, megaureter in two, hypoplastic kidney in four, vesicoureteral reflux in six (three were accompanied by hypoplastic kidneys or multicystic dysplastic kidney), multicystic dysplastic kidney in one, and horseshoe kidney in one.

Conclusion:
These results indicate that our screening methods and criteria are useful variables for detecting congenital kidney and urinary tract abnormalities.