bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of a colour pattern: history, development, and selection
Authors: Price T. , Pavelka M.
Published: December 2004

Patterns of melanin pigmentation in birds are extremely varied. Nevertheless it is easy to think of many patterns that are never observed, and others that frequently recur in diverse and distantly related species. Using as our model the avian genus Phylloscopus we ask how the restricted range of observed patterns might be attributable to a restricted range of variants produced by developmental perturbations. The patterns we consider consist of unmelanized patches on the wings, crown and rump on otherwise pigmented upperparts. We use reaction‐diffusion models to show that gross features of the pattern can be simply predicted from considerations of embryo shape. We suggest that birds are expected to have more patterned heads, because the head region is relatively larger than other regions in the developing embryo. A comparative analysis across many species of birds and a phylogenetic analysis within the genus Phylloscopus show that the component elements of the pattern have repeatedly been lost and gained during evolution. A shift in a threshold reading could explain the appearance and disappearance of the unmelanized patches, perhaps through changes in the sensitivity of melanocytes to epidermal signals. Such threshold shifts would make the transition between patterned and unpatterned forms particularly easy once the patterns have been exposed to selection in some distant ancestor. This partitioning of the roles of selection and development implies that many features of the patterns reflect developmental mechanisms in both immediate and more distant ancestors.
Frequency‐dependent selection by pollinators: mechanisms and consequences with regard to behaviour of bumblebees Bombus terrestris (L.) (Hymenoptera: Apidae)
Authors: Smithson A. , Macnair M. R.
Published: December 2004

Bombus terrestris , a typical pollinating insect species, was offered artificial flowers of two different corolla colours with the same sucrose solution reward in an array. Common colours were significantly preferred, and the strength of the frequency‐dependent response increased as a result of learning. There were also frequency‐independent biases towards blue flowers, probably because blue flowers appeared more conspicuous to bumblebees than yellow flowers, and the degree of preference for blue was greater when flowers had low nectar rewards. Flower‐to‐flower movements by individual bumblebees between flowers were non‐random, were biased to movements within the same flower colour, and were also dependent on morph frequency. The mechanisms governing flower selection in bumblebees are discussed.

Pollinators foraging similarly in a natural situation would induce positive frequency‐dependent selection, assortative mating, and directional selection on different corolla colour morphs of the plant population being visited, resulting in stabilizing selection for a single flower colour.
The evolution of the reptilian hindfoot and the homology of the hooked fifth metatarsal
Authors: Lee M. S. Y.
Published: December 2004

The homology of the fifth metatarsal in turtles and in diapsid reptiles is reassessed in the light of new phylogenetic studies. The two nearest outgroups to turtles — pareiasaurs and procolophonoids — both lack a fifth distal tarsal but retain a normal fifth metatarsal. The fifth distal tarsal was therefore lost at the base of the clade that contains turtles. Thus, the hooked fifth metatarsal in turtles must consist entirely of a modified fifth metatarsal: it does not include contributions from the fifth distal tarsal, as commonly supposed. In diapsids, loss of the fifth distal tarsal appears to have occurred at the base of crown‐clade diapsids, hooking of the fifth metatarsal subsequently occurring within lepidosauromorphs, and in archosauromorphs. If so, the hooked fifth metatarsal in archosauromorphs, and some lepidosauromorphs, consists entirely of a modified fifth metatarsal. In both turtles and diapsids, integration of the elements distal to the mesotarsal joint precedes evolution of the hooked fifth metatarsal, supporting the view that the latter element evolved to perform a lever function (analogous to the “heel bone” of mammals).
Phenotypic plasticity and selection in Drosophila life history evolution. 2. Diet, mates and the cost of reproduction
Authors: Chippindale A. K. , Leroi A. M. , Saing H. , Borash D. J. , Rose M. R.
Published: December 2004

While it is commonplace for biologists to use the response to environmental manipulation as a guide to evolutionary responses to selection, the relationship between phenotypic plasticity and genetic change is not generally well‐established. The life‐histories of laboratory Drosophila populations are among the few experimental systems which simultaneously afford information on phenotypic plasticity and evolutionary trajectories. We employed a combination of two replicated selectively differentiated stocks (postponed aging stocks and their controls; 10 populations in total) and two different environmental manipulations (nutrition and mating) to explore the empirical relationship between phenotypic plasticity and evolutionary trajectories. While there are a number of parallels between the results obtained using these two approaches, there are important differences. In particular, as the detail of the biological characterization of either type of response increases, so their disparities multiply. Nonetheless, the combination of environmental manipulation with evolutionary divergence provides valuable information about the biological connections between life‐history, caloric reserves, and reproductive physiology in Drososphila.
Evolution of animal genitalia: morphological correlates of fitness components in a water strider
Authors: Arnqvist G. , Thornhill R. , Rowe L.
Published: December 2004

Rapid divergence of male genitalia is one of the most general evolutionary trends in animals with internal fertilization, but the mechanisms of genital evolution are poorly understood. The current study represents the first comprehensive attempt to test the main hypotheses that have been suggested to account for genital evolution (the lock‐and‐key, sexual selection and pleiotropy hypotheses) with intraspecific data. We measure multivariate phenotypic selection in a water strider species, by relating five different components of fitness (mating frequency, fecundity, egg hatching rate, offspring survival rate and offspring growth rate) to a suite of genital and non‐genital morphological traits (in total 48). Body size had a series of direct effects in both sexes. Large size in females was positively related to both fecundity and egg hatching rate. There was positive sexual selection for large size in males (mating frequency), which to some extent was offset by a reduced number of eggs laid by females mated to large males. Male genitalic morphology influenced male mating frequency, but the detected directional selection on genitalia was due to indirect selection on phenotypically correlated non‐intromittent traits. Further, we found no assortative mating between male intromittent genitalia and female morphology. Neither did we find any indications of male genitalia conveying information of male genetic quality. Several new insights can be gained from our study. Most importantly, our results are in stark disagreement with the long standing lock‐and‐key hypothesis of genital evolution, as well as with certain models of sexual selection. Our results are, however, in agreement with other models of sexual selection as well as with the pleiotropy hypothesis of genital evolution. Fluctuating asymmetry of bilaterally symmetrical traits, genital as well as non‐genital, had few effects on fitness. Females with low fluctuating asymmetry in leg length produced offspring with a higher survival rate, a pattern most proba bly caused by direct phenotypic maternal effects. We also discuss the relevance of our results to sexual conflict over mating, and the evolution of sexual traits by coevolutionary arms races between the sexes.
The molecular basis of dominance relationships: the case of some recent adaptive genes
Authors: Bourguet D. , Raymond M.
Published: December 2004

A controversial debate in evolutionary biology has been to explain why deleterious mutations are usually recessive to their wild‐type allele. For Fisher, dominance of the wild‐type is the result of selection, whereas for Wright it is a mere consequence of the biochemical properties of physiological pathways. Over time, Wright's theory has appeared as the most appropriate, and Kacser and Burns explained why the widespread occurrence of recessive mutants is the inevitable consequence of the kinetic structure of enzyme networks. Does Wright and Kacser and Burns (W‐K‐B)'s theory apply for newly arisen adaptive genes? A survey of more than 70 studies shows that pesticide resistance conferred by mutations decreasing the affinity of the pesticide target‐sites varies from complete recessivity to complete dominance. This review shows that dominance always has a purely physiological explanation that can be roughly, but not simply, predicted by W‐K‐B's theory. Thus, although W‐K‐B's theory remains powerful for predicting the recessivity of deleterious mutations involved in enzymatic pathways, no general theory emerges from the study of other situations, and molecular explanations are to be sought on a case by case basis.
Epigenetic inheritance in evolution
Authors: Jablonka E. , Lamb M. J.
Published: December 2004

We discuss the role of cell memory in heredity and evolution. We describe the properties of the epigenetic inheritance systems (EISs) that underlie cell memory and enable environmentally and developmentally induced cell phenotypes to be transmitted in cell lineages, and argue that transgenerational epigenetic inheritance is an important and neglected part of heredity. By looking at the part EISs have played in the evolution of multicellularity, ontogeny, chromosome organization, and the origin of some post‐mating isolating mechanisms, we show how considering the role of epigenetic inheritance can sometimes shed light on major evolutionary processes.
Loss of genetic diversity and fitness in Common Toad (Bufo bufo) populations isolated by inimical habitat
Authors: Hitchings S. P. , Beebee T. J. C.
Published: December 2004

Measures of genetic diversity (including heterozygosity), survival and developmental homeostasis were found to be significantly lower in small, urban populations of the Common Toad (Bufo bufo) than in larger, rural populations of the same region. The autecology and genetic analysis of this relatively sedentary species suggested that the causal mechanism was genetic drift, arising from barriers to migration created by urban development. The pre‐metamorphic survival of larvae cultured in identical conditions increased positively with the mean number of alleles at a locus and the percentage of polymorphic loci. Observed heterozygosity in urban garden and rural populations was correlated inversely with the number of observed physical abnormalities (used as a measure of developmental homeostasis) in the developing tadpoles. Genetic distances between town sites of mean 2.2 km separation were significantly higher than those between rural sites of mean 37 km separation. Genetic data were based on allozyme analysis of 27 loci in 8 urban and 4 rural populations. A subset of these sites (3 urban, 2 rural) were also assessed at 3 minisatellite loci and a positive correlation found between the average number of alleles per locus detected by the two methods. Estimates of Nei's 1972 genetic distance, derived separately from the DNA and protein data, were not, however, correlated. The reduction in genetic diversity and fitness observed in these urban toads provides an example of the effect on population persistence that longer term depletion in numbers and habitat fragmentation can have in the wider environment.
Transcription termination at the mouse mitochondrial H‐strand promoter distal site requires an A/T rich sequence motif and sequence specific DNA binding proteins
Authors: Camasamudram Vijayasarathy , Fang Ji‐Kang , Avadhani Narayan G.
Published: December 2004

Termination of mitochondrial (mt) H‐strand transcription in mammalian cells occurs at two distinct sites on the genome. The first site of termination, referred to as mt‐TERM occurs beyond the 16 S rRNA gene. However, the second and final site of termination beyond the tRNAThr gene remains unclear. In this study we have characterized the site of termination of the polycistronic distal gene transcript beyond the D‐loop region, immediately upstream of the tRNAPhe gene. This region, termed D‐TERM, maps to nucleotides 16274–16295 of the mouse genome and includes a conserved A/T rich sequence motif AATAAA as a part of the terminator. Gel‐shift analysis showed that the 22 bp D‐TERM DNA forms two major complexes with mouse liver mt extract in a sequence‐specific manner. Protein purification by DNA‐affinity chromatography yielded two major proteins of 45 kDa and 70 kDa. Finally, the D‐TERM DNA can mediate transcription termination in a unidirectional manner in a HeLa mt transcription system, only in the presence of purified mouse liver mt D‐TERM DNA binding proteins. We have therefore characterized a novel mt transcription termination system, similar in some properties to that of sea urchin, as well as the nuclear RNA Pol I and Pol II transcription termination systems.
Disease‐related mutations in cytochrome c oxidase studied in yeast and bacterial models
Authors: Bratton Melyssa , Mills Denize , Castleden C. Kate , Hosler Jonathan , Meunier Brigitte
Published: December 2004

Mitochondrial cytochrome c oxidase is a key protonmotive component of the respiratory chain. Mutations in the mitochondrially‐encoded subunits of the complex have been reported in association with a range of diseases. In this work we used yeast and bacterial mutants to assess the effect of human mutations in subunit 1 (L196I) and subunit 3 (G78S, A200T, ΔF94–F98, F251L and W249Stop). While the stop mutation at the C‐terminus of subunit 3 and the short deletion were highly deleterious and abolished the assembly of the mitochondrial enzyme, the four missense mutations caused little or no effect on the respiratory function. Detailed analysis of G78S, A200T and ΔF94–F98 in Rhodobacter sphaeroides confirmed and extended these observations. We show in this study that the combination of yeast and bacterial models is a useful tool to elucidate the effect of mutations in the catalytic core of cytochrome oxidase. The yeast enzyme is highly similar to the human enzyme and provides a good model to assess the deleterious effect of reported mutations. The bacterial system allows detailed biochemical analysis of the effect of the mutations on the function and assembly of the catalytic core of the enzyme.
Adaptability and flexibility of HIV‐1 protease
Authors: Kumar Mukesh , Hosur Madhusoodan V.
Published: December 2004

Even though more than 200 three‐dimensional structures of HIV‐1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few structures of unliganded protein have been determined. We have recently solved structures of unliganded HIV‐1 protease tethered dimer mutants to resolutions of 1.9 Å and 2.1 Å, and have found that the flaps assume closed‐flap conformation even in the absence of any bound ligand. We report comparison of the unliganded closed‐flap structure with structures of HIV‐1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV‐1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV‐1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexibility and adaptability of proteins in general.
Plasmoredoxin, a novel redox‐active protein unique for malarial parasites
Authors: Becker Katja , Kanzok Stefan M. , Iozef Rimma , Fischer Marina , Schirmer R. Heiner , Rahlfs Stefan
Published: December 2004

Thioredoxins are a group of small redox‐active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development.

Here we describe the identification and characterization of a novel 22 kDa redox‐active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin‐reduction assay and reduces glutathione disulfide with a rate constant of 640 m−1·s−1 at pH 6.9 and 25 °C; glutathione‐dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood‐stage forms of malarial parasites.

Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.
Steady‐state and time‐resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli
Authors: Polit Agnieszka , Błaszczyk Urszula , Wasylewski Zygmunt
Published: December 2004

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady‐state and time‐resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5‐I‐AEDANS attached to Cys178. Binding of cAMP in the CRP–(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP–(cAMP)4 complex alters the surroundings of Trp85. Time‐resolved anisotropy measurements indicated that cAMP binding in the CRP–(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP–(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 Å in apo‐CRP to 18.7 Å in the CRP–(cAMP)2 complex. The binding of cAMP in the CRP–(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP–DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to ≈ 20 Å. The spectral changes observed provide new structural information about the cAMP‐induced allosteric activation of the protein.
Mitochondrial connection to the origin of the eukaryotic cell
Authors: Emelyanov Victor V.
Published: December 2004

Phylogenetic evidence is presented that primitively amitochondriate eukaryotes containing the nucleus, cytoskeleton, and endomembrane system may have never existed. Instead, the primary host for the mitochondrial progenitor may have been a chimeric prokaryote, created by fusion between an archaebacterium and a eubacterium, in which eubacterial energy metabolism (glycolysis and fermentation) was retained. A Rickettsia‐like intracellular symbiont, suggested to be the last common ancestor of the family Rickettsiaceae and mitochondria, may have penetrated such a host (pro‐eukaryote), surrounded by a single membrane, due to tightly membrane‐associated phospholipase activity, as do present‐day rickettsiae. The relatively rapid evolutionary conversion of the invader into an organelle may have occurred in a safe milieu via numerous, often dramatic, changes involving both partners, which resulted in successful coupling of the host glycolysis and the symbiont respiration. Establishment of a potent energy‐generating organelle made it possible, through rapid dramatic changes, to develop genuine eukaryotic elements. Such sequential, or converging, global events could fill the gap between prokaryotes and eukaryotes known as major evolutionary discontinuity.
Association of feather colour with constitutively active melanocortin 1 receptors in chicken
Authors: Ling Maria K. , Lagerström Malin C. , Fredriksson Robert , Okimoto Ronald , Mundy Nicholas I. , Takeuchi Sakae , Schiöth Helgi B.
Published: December 2004

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e+R, e+B&D, ewh/ey, ERfayoumi gave receptors to which melanocortin stimulating hormone (α‐MSH) and NDP‐MSH bound with similar IC50 values and responded to α‐MSH by increasing intracellular cAMP levels in a dose‐dependent manner. Three of the cMC1 receptors; eb, E and ER, did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and ER alleles are associated with black feather colour in chicken while the eb allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1‐E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4‐E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the ‘E92K switch’ mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.
Functional fine‐mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein
Authors: Pütz Mike M. , Hoebeke Johan , Ammerlaan Wim , Schneider Serge , Muller Claude P.
Published: December 2004

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine‐map the highly conserved hemagglutinin noose epitope (H379–410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus‐neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729–735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild‐type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent‐exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent‐hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.
The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC
Authors: Facey Sandra J. , Kuhn Andreas
Published: December 2004

KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions. The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop. The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD. To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region. The results show that also the first loop was translocated independently of YidC and the Sec translocase. We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases.

When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC. To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal‐sized fragments. Both constructed fragments, which contained KdpD‐N (residues 1–448 of KdpD) and the KdpD‐C (residues 444–894 of KdpD), readily inserted into the membrane. Similar to the epitope‐tagged KdpD protein, only KdpD‐C depended on the presence of the Sec translocase and YidC. This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.
Functional analysis of disease‐causing mutations in human galactokinase
Authors: Timson David J. , Reece Richard J.
Published: December 2004

Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild‐type enzyme. One mutant (A198V) had kinetic properties that were essentially wild‐type. These results are discussed both in terms of galactokinase structure‐function relationships and how these functional changes may relate to the causes of galactosemia.
Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses
Authors: Borowski Peter , Deinert Johanna , Schalinski Sarah , Bretner Maria , Ginalski Krzysztof , Kulikowski Tadeusz , Shugar David
Published: December 2004

A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)‐associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low‐molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7‐tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC50 is approximately 20 µm with a DNA substrate, but only 60 µm with an RNA substrate. Several related analogues of TBBT were enzyme‐ and/or substrate‐specific inhibitors. For example, 5,6‐dichloro‐1‐(β‐d‐ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC50 ≈ 0.3 µm), but much weaker with a DNA substrate (IC50 ≈ 3 µm). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 µm. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,‐tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP‐binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP‐binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.
The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase
Authors: Miertzschke Mandy , Greiner‐Stöffele Thomas
Published: December 2004

A thermostable enzyme from the hyperthermophilic sulphate‐reducing archaeon, Archaeoglobus fulgidus, was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli. Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III. The 774 bp long gene was isolated from a culture sample and cloned into different vectors. Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU, ileY and leuW tRNA genes as a host strain. The lack of thioredoxin reductase (trxB) and glutathione reductase (gor) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol. Purification was performed by heat treatment of the soluble fraction at 80 °C for 30 min followed by a two‐step ion exchange chromatography. The activity of the enzyme could be maintained. Optimal activity was achieved at 80 °C and at a pH of 7. Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg2+ as described [Ankenbauer, W., Laue, F., Sobek, H., & Greif, M. (2000), patent number WO2001023583]. Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed. In contrast to exonuclease III from Escherichia coli, the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3′‐termini of linear double stranded DNA. The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg2+ with Ca2+ ions.
Antisense technologies
Authors: Kurreck Jens
Published: December 2004

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence‐specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti‐mRNA strategies can be distinguished. Firstly, the use of single stranded antisense‐oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side‐effects. Some of these problems have been solved in ‘second generation’ nucleotides with alkyl modifications at the 2′ position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA‐cleaving ribozymes and deoxyribozymes, and the use of 21‐mer double‐stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.
Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom
Authors: Nicastro Giuseppe , Franzoni Lorella , de Chiara Cesira , Mancin Adriana C. , Giglio Josè R. , Spisni Alberto
Published: December 2004

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross‐linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 µg per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage‐sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N‐terminal α‐helix and a small antiparallel triple‐stranded β‐sheet arranged in an αβ1β2β3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an α/β fold, though with a β1αβ2β3 topology.

In addition, as the antibacterial β‐defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human β‐defensins shows a similar fold and a comparable net positive potential surface.

To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.
Involvement of the V2 receptor in vasopressin‐stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells
Authors: Masuda Shinako , Hattori Akira , Matsumoto Hideko , Miyazawa Shinobu , Natori Yasuhiro , Mizutani Shigehiko , Tsujimoto Masafumi
Published: December 2004

The placental leucine aminopeptidase (P‐LAP)/oxytocinase is a membrane‐bound enzyme thought to play an important role during pregnancy. In this study, we identified the presence of P‐LAP protein in the renal distal tubules and collecting ducts. In rat NRK52E cells derived from renal tubules, P‐LAP was localized mainly in the intracellular compartment. Upon the treatment of cells with 8‐arginine‐vasopressin (AVP), a significant increase in the surface level of P‐LAP was observed. [deamino‐Cys1, d‐Arg8]‐vasopressin (DDAVP), a specific V2 receptor agonist, increased the surface level of P‐LAP, while [adamantaneacetyl1, O‐Et‐d‐Tyr2, Val4, aminobutyryl6, Arg8,9]‐vasopressin (AEAVP), a potent V2 receptor antagonist, blocked the AVP‐stimulated enhancement. Moreover, reagents known to enhance the intracellular level of cAMP have also been shown to increase the surface level of P‐LAP. When we examined the colocalization of P‐LAP with the cell surface water channel aquaporin‐2 (AQP‐2) that is regulated by AVP, the P‐LAP‐containing vesicles had a relatively higher density than the AQP‐2‐containing vesicles, suggesting that P‐LAP and AQP‐2 are differently distributed in NRK52E cells. These results suggest that AVP induces the translocation of P‐LAP via V2 receptor and P‐LAP plays a role in the regulation of excessive AVP level in the renal collecting duct, acting as a negative feedback mechanism for the AVP action of regulating water reabsorption.
Saccharomyces cerevisiae Pip2p–Oaf1p regulates PEX25 transcription through an adenine‐less ORE
Authors: Rottensteiner Hanspeter , Hartig Andreas , Hamilton Barbara , Ruis Helmut , Erdmann Ralf , Gurvitz Aner
Published: December 2004

The role of the Saccharomyces cerevisiae Pip2p–Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation. The PEX25 promoter contains an oleate response element (ORE)‐like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p–Oaf1p binding. Pex25p levels were higher in wild‐type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p–Oaf1p. Studies based on lacZ reporter genes and in vitro protein–DNA interactions revealed that the PEX25 ORE could bind Pip2p–Oaf1p and confer activation on a basal promoter. These findings reinforced the central role played by Pip2p–Oaf1p in regulating peroxisome proliferation. We also investigated whether Pip2p–Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix. Pip2p–Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE‐like CGG palindrome in the PEX14 promoter. However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol. These data on a functional, adenine‐less, PEX25 ORE and a nonfunctional N13‐spaced ORE‐like sequence in the PEX14 promoter capable of binding Pip2p–Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8−12CCG.
Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules
Authors: Contín María A. , Purro Silvia A. , Bisig C. Gastón , Barra Héctor S. , Arce Carlos A.
Published: December 2004

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor‐okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.
Functional assembly of thylakoid ΔpH‐dependent/Tat protein transport pathway components in vitro
Authors: Fincher Vivian , Dabney‐Smith Carole , Cline Kenneth
Published: December 2004

Assembly of the components of the thylakoid ΔpH‐dependent/Tat protein transport machinery was analyzed in vitro. Upon incubation with intact chloroplasts, precursors to all three components, Hcf106, cpTatC and Tha4, were imported into the organelle and assembled into characteristic endogenous complexes. In particular, all of the imported cpTatC and approximately two‐thirds of the imported Hcf106 functionally assembled into 700 kDa complexes capable of binding Tat pathway precursor proteins. The amounts assembled into thylakoids by this procedure were moderate. However, physiological quantities of mature forms of Tha4 and Hcf106 were integrated into isolated thylakoids and a significant percentage of the Hcf106 so integrated was assembled into the 700 kDa complex. Interestingly, a mutant form of Hcf106 in which an invariant transmembrane glutamate was changed to glutamine integrated into the membrane but did not assemble into the receptor complex. Analysis of energy and known pathway component requirements indicated that Hcf106 and Tha4 integrate by an unassisted or ‘spontaneous’ mechanism. The functionality of in vitro integrated Tha4 was verified by its ability to restore transport to thylakoid membranes from the maize tha4 mutant, which lacks the Tha4 protein. Development of this functional in vitro assembly assay will facilitate structure–function studies of the thylakoid Tat pathway translocation machinery.
The Effects of Starvation on Crayfish Responses to Alarm Odor
Authors: Hazlett Brian A.
Published: December 2004

The effect of starvation on responses to alarm odor was tested with individuals of an invasive and a native species of crayfish. I predicted that chemical predator cues would inhibit feeding less in starving than well‐fed animals, and that this decrease would be stronger in a native compared with an invasive species. Individuals were exposed to food odor and then alarm odor after 3 and 10 d of starvation. The inhibition of food‐related behavior patterns was similar on the 2 d of testing for individuals of the invasive species, Orconectes rusticus. Individuals of O. virilis showed a significant reduction in the effects of alarm odor detection on day 10 compared with day 3 of starvation. The lack of a change in responses to alarm odors between days of testing by individuals of O. rusticus may be because they are more responsive to alarm odors than individuals of O. virilis. This behavioral difference could contribute to the successful range extension of O. rusticus.
Productive phenology of tropical montane forests: Fertilization experiments along a moisture gradient
Authors: Nomura Naofumi , Kikuzawa Kihachiro
Published: December 2004

Phenological responses of leaves and roots were studied in the tropical montane forests of Mount Kinabalu, Borneo. Soil nutrient supply, in addition to the supply of light and water, is a potential abiotic factor influencing plant phenology in the tropics. The main objective of this study was to evaluate the contribution of soil nutrient supply to plant productive phenology. Fertilization experiments, including controls, nitrogen fertilized and nitrogen and phosphorus fertilized treatments, were conducted on three vegetation types in different moisture environments. Responses of leaves and roots were compared among treatments and among vegetation types. Leaf flushing was induced by nitrogen fertilization in the upper montane forest, where extremely wet moisture conditions are associated with cloud cover. This induction of leaf flushing by fertilization was not observed in the other forests. Root growth was suppressed by fertilization when leaf flushing was not induced by fertilization. These results indicate that soil pulsed nutrient release could be a cue for leaf flushing in a tropical wet environment, and that leaf phenology could be regulated by external abiotic factors and root phenology could be regulated by internal plant demands.
Restricted seed contribution and clonal dominance in a free‐floating aquatic plant Utricularia australis R. Br. in southwestern Japan
Authors: Araki Satoru , Kadono Yasuro
Published: December 2004

Utricularia australis R. Br. is an aquatic angiosperm species common in natural and irrigation ponds in temperate regions. This species reproduces both sexually and vegetatively, but in southwestern Japan the occurrence of male‐sterile populations, in which plants produce no pollen and propagate only vegetatively, has been recorded. We studied the reproductive contribution of seeds in normal pollen‐producing populations using isozyme analyses, a pollination experiment under culture and field observations. Although seedlings obtained from controlled mating indicated segregation of isozyme, polymorphism of the isozyme genotype was detected mainly among populations, but rarely within each pond population. This suggested clonal dominance and rarity of seed or seedling survival in natural populations. In the pollination experiment, the mean seed set ratio in cross‐pollination between plants of the same isozyme genotype (7.6%) did not differ significantly from self‐pollination (7.6%), but was lower than cross‐pollination between plants of different genotypes (45.7%). The low ratio in crossing between the same genotype plants was ascribed to the clonality of the parents. In general, these results corresponded with the low ratios in seed setting observed in natural populations (7.9–13.7%). All the male‐sterile populations we surveyed showed the same genotype, thus male sterility in the study area was considered to have the same origin.
Impact of blood testing on patient disposition from the emergency department
Authors: Knott Jonathan C , Meyer Alastair D McR
Published: December 2004

Objective: To determine whether blood test results lead to a change in planned disposition of patients from the ED.

Methods: A prospective review of the anticipated and actual disposition of patients from the ED before and after blood test results, including stratification by seniority of medical staff.

Results: There were 256 patients enrolled, 53% had blood tests requested. Expected disposition was not altered by test results in 87% of patients having blood tests. Medical staff were poor at identifying those patients whose disposition would be altered by their blood results (sensitivity 44%, specificity 72%, negative predictive value 89%). Seniority of medical staff was not associated with an alteration in disposition due to the blood results (P = 0.37).

Conclusions: Blood test results have minimal impact on expected patient disposition from the ED. Recognition of this may improve resource utilisation with earlier discharge planning.