bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of intestinal parasites in rural Southern Indians
Authors: Kang Gagandeep , Mathew Mary S. , Prasanna Rajan D. , Daniel Jasper D. , Mathan Minnie M. , Mathan V. I. , Muliyil J. P.
Published: June 2003

objective
 
 To determine the prevalence of intestinal protozoal and helminthic infection in a rural population.method  Seventy‐eight members of 15 families from a village were studied. Stool samples from all subjects were examined on alternate days for one month.results  The overall prevalence rate of various parasitic infections was 97.4%, with only 2 of 78 subjects not excreting parasites in any of their 15 samples. Eighteen (23.1%) persons had only one type of parasite, while 58 (74.3%) excreted multiple parasites. Giardia and Cryptosporidium were the commonest protozoan infections, affecting 42/78 (53.8%) and 31/78 (39.7%), respectively. Hookworm infestations were the commonest helminthic infections, seen in 48/78 (61.5%). Based on excretion patterns, the asymptomatic individuals could be divided into 2 groups of infrequent and frequent excretors, indicating that the host response may determine the level of parasite replication in the gut.
Anthropogenic disturbance promotes hybridization between Banksia species by altering their biology
Authors: Lamont B. B. , He T. , Enright N. J. , Krauss S. L. , Miller B. P.
Published: June 2003

Putative hybrids between Banksia hookeriana and B. prionotes were identified among 12 of 106 populations of B. hookeriana located at or near anthropogenically disturbed sites, mainly roadways, but none in 156 undisturbed populations. Morphometrics and AFLP markers confirmed that a hybrid swarm existed in a selected disturbed habitat, whereas no intermediates were present where the two species co-occurred in undisturbed vegetation. Individuals of both species in disturbed habitats at 12 sites were more vigorous, with greater size and more flower heads than their counterparts in undisturbed vegetation. These more fecund plants also showed a shift in season and duration of flowering. By promoting earlier flowering of B. hookeriana plants and prolonging flowering of B. prionotes, anthropogenic disturbance broke the phenological barrier between these two species. We conclude that anthropogenic disturbance promotes hybridization through increasing opportunities for gene flow by reducing interpopulation separation, increasing gamete production and, especially, promoting coflowering.
Evolvability and genetic constraint in Dalechampia blossoms: components of variance and measures of evolvability
Authors: Hansen Thomas F. , Pélabon Christophe , Armbruster W. Scott , Carlson Matthew L.
Published: June 2003

Many evolutionary arguments are based on the assumption that quantitative characters are highly evolvable entities that can be rapidly moulded by changing selection pressures. The empirical evaluation of this assumption depends on having an operational measure of evolvability that reflects the ability of a trait to respond to a given external selection pressure. We suggest short-term evolvability be measured as expected proportional response in a trait to a unit strength of directional selection, where strength of selection is defined independently of character variation and in units of the strength of selection on fitness itself. We show that the additive genetic variance scaled by the square of the trait mean, IA, is such a measure. The heritability, h2, does not measure evolvability in this sense. Based on a diallel analysis, we use IA to assess the evolvability of floral characters in a population of the neotropical vine Dalechampia scandens (Euphorbiaceae). Although we are able to demonstrate that there is additive genetic variation in a number of floral traits, we also find that most of the traits are not expected to change by more than a fraction of a percent per generation. We provide evidence that the degree of among-population divergence of traits is related to their predicted evolvabilities, but not to their heritabilities.
Sexual selection promotes hybridization between Pecos pupfish, Cyprinodon pecosensis and sheepshead minnow, C. variegatus
Authors: Rosenfield J. A. , Kodric-Brown A.
Published: June 2003

Rapid and extensive genetic introgression has occurred between Pecos pupfish (Cyprinodon pecosensis) and sheepshead minnow (Cyprinodon variegatus) in the wild. We studied both female mate choice and male–male competition for mates among C. pecosensis, C. variegatus, and their F1 hybrids to determine what role these behaviours played in the formation of the hybrid swarm. Female C. pecosensis preferred male C. variegatus to conspecific males, C. variegatus females displayed no significant preference when given a choice between purebred males, and neither C. pecosensis nor C. variegatus females discriminated against F1 hybrid males. We found no evidence for female olfactory recognition of mates. Male F1 hybrids and C. variegatus were more aggressive than C. pecosensis males, achieving greater reproductive success under two different experimentally-induced mating systems. Hybrids were superior to C. variegatus when only two males competed (dominance interactions), but the two types were competitively equivalent in a territorial mating system. Our results indicate that active inter- and intra-sexual selection contributed to the accelerated hybridization between these two species. By including the possibility that some aspects of a hybridization and introgression event may be under positive selection, researchers may better understand the dynamics that lead to hybrid zone stability or the spread of introgressed genetic material.
Local mate competition with lethal male combat: effects of competitive asymmetry and information availability on a sex ratio game
Authors: Abe J. , Kamimura Y. , Ito H. , Matsuda H. , Shimada M.
Published: June 2003

We constructed a sex allocation model for local mate competition considering the asymmetry of competitive abilities among sons. This model assumes two females of a parasitoid wasp oviposit on the same host in sequential order. The evolutionarily stable strategy will be in either Stackelberg or Nash equilibrium, depending on whether the females can recognize their opponent's sex ratio or not, respectively. The Nash equilibrium predicts the second female produce more males than the first. If the second female is able to know and respond to the strategy of the first (a Stackelberg equilibrium), the first will decide an optimal sex ratio assuming that the second reply to it. Under such an assumption, our model predicts that not producing sons is adaptive for the second female when the sons she produces have low competitive ability. Males of parasitoid wasps Melittobia spp. are engaged in lethal male–male combat, indicating large asymmetry in mating success among sons. If females have the ability to recognize their opponent's sex ratio, our model suggests that the severe lethal male–male combat may be one factor explaining their extremely female-biased sex ratio that is unexplainable by pre-existent models.
Selective and genetic constraints on the evolution of body size in a stream-dwelling salmonid fish
Authors: Wilson A. J. , Hutchings J. A. , Ferguson M. M.
Published: June 2003

To examine constraints on evolution of larger body size in two stunted populations of brook charr (Salvelinus fontinalis) from a single river in Cape Race, Newfoundland, Canada, we measured viability selection acting on length-at-age traits, and estimated quantitative genetic parameters in situ (following reconstruction of pedigree information from microsatellite data). Furthermore we tested for phenotypic differentiation between the populations, and for association of high juvenile growth with early maturity that is predicted by life history theory. Within each population, selection differentials and estimates of heritabilities for length-at-age traits suggested that evolution of larger size is prevented by both selective and genetic constraints. Between the populations, phenotypic differentiation was found in length-at-age and age of maturation traits, whereas early maturation was associated with increased juvenile growth (relative to adult growth) both within and between populations. The results suggest an adaptive plastic response in age of maturation to juvenile growth rates that have a largely environmental basis of determination.
The evolution of plumage polymorphism in birds of prey and owls: the apostatic selection hypothesis revisited
Authors: Fowlie M. K. , Krüger O.
Published: June 2003

Co-evolution between phenotypic variation and other traits is of paramount importance for our understanding of the origin and maintenance of polymorphism in natural populations. We tested whether the evolution of plumage polymorphism in birds of prey and owls was supported by the apostatic selection hypothesis using ecological and life-history variables in birds of prey and owls and performing both cross taxa and independent contrast analyses. For both bird groups, we did not find any support for the apostatic selection hypothesis being the maintaining factor for the polymorphism: plumage polymorphism was not more common in taxa hunting avian or mammalian prey, nor in migratory species. In contrast, we found that polymorphism was related to variables such as sexual plumage dimorphism, population size and range size, as well as breeding altitude and breeding latitude. These results imply that the most likely evolutionary correlate of polymorphism in both bird groups is population size, different plumage morphs might simply arise in larger populations most likely because of a higher probability of mutations and then be maintained by sexual selection.
Sexual selection, antennae length and the mating advantage of large males in Asellus aquaticus
Authors: Bertin A. , Cezilly F.
Published: June 2003

In crustacean species with precopulatory mate-guarding, sexual size dimorphism has most often been regarded as the consequence of a large male advantage in contest competition for access to females. However, large body size in males may also be favoured indirectly through scramble competition. This might partly be the case if the actual target of selection is a morphological character, closely correlated with body size, involved in the detection of receptive females. We studied sexual selection on body size and antennae length in natural populations of Asellus aquaticus, an isopod species with precopulatory mate guarding. In this species, males are larger than females and male pairing success is positively related to body size. However, males also have longer antennae, relative to body size, than females, suggesting that this character may also be favoured by sexual selection. We used multivariate analysis of selection to assess the relative influences of body size and antennae length in five different populations in the field. Selection gradients indicated that overall body size was a better predictor of male pairing success than antennae length, although some variation was observed between sites. We then manipulated male antennae length in a series of experiments conducted in the laboratory, and compared the pairing ability of males with short or long antennae. Males with short antennae were less likely to detect, orient to and to pair with a receptive female compared to males with long antennae. We discuss the implications of our results for studies of male body size and sexual dimorphism in relation to sexual selection in crustaceans.
The evolution of inquilinism, host-plant use and mitochondrial substitution rates in Tamalia gall aphids
Authors: Miller D. G. , Crespi B.
Published: June 2003

We used mitochondrial DNA data to infer phylogenies for 28 samples of gall-inducing Tamalia aphids from 12 host-plant species, and for 17 samples of Tamalia inquilinus, aphid ‘inquilines’ that obligately inhabit galls of the gall inducers and do not form their own galls. Our phylogenetic analyses indicate that the inquilines are monophyletic and closely related to their host aphids. Tamalia coweni aphids from different host plants were, with one exception, very closely related to one another. By contrast, the T. inquilinus aphids were strongly genetically differentiated among most of their host plants. Comparison of branch lengths between the T. coweni clade and the T. inquilinus clade indicates that the T. inquilinus lineage evolves 2.5–3 times faster for the cytochrome oxidase I gene. These results demonstrate that: (1) Tamalia inquilines originated from their gall-inducing hosts, (2) communal (multi-female) gall induction apparently facilitated the origin of inquilinism, (3) diversification of the inquilines has involved rapid speciation along host-plant lines, or the rapid evolution of host-plant races, and (4) the inquilines have undergone accelerated molecular evolution relative to their hosts, probably due to reduced effective population sizes. Our findings provide insight into the behavioural causes and evolutionary consequences of transitions from resource generation to resource exploitation.
Do precocial mammals develop at a faster rate? A comparison of rates of skull development in Sigmodon fulviventer and Mus musculus domesticus
Authors: Zelditch Miriam Leah , Lundrigan Barbara L. , David Sheets H. , Garland Theodore
Published: June 2003

Variation in neonatal maturity among mammals is often explained by variation in gestation length, but species may also differ in developmental rate, a quantity that is difficult to measure because the conventional formalism makes two important and potentially unrealistic assumptions: (1) ontogeny of form can be described by a single line, and (2) species have the same ontogeny of form. We examine two species, one precocial (Sigmodon fulviventer), the other altricial (Mus musculus domesticus), and find that neither assumption is met. Therefore, we introduce an alternative metric, the rate of shape differentiation away from the average neonate. We find that S. fulviventer has a lower developmental rate than M. m. domesticus; consequently, while more mature at birth, S. fulviventer loses ground to M. m. domesticus over time. Surprisingly, despite differences in gestation length and developmental rate, these species reach developmental and life-history milestones at nearly identical degrees of skull shape maturity.
Colour polymorphism in birds: causes and functions
Authors: Galeotti P. , Rubolini D. , Dunn P. O. , Fasola M.
Published: June 2003

We studied polymorphism in all species of birds that are presently known to show intraspecific variation in plumage colour. At least three main mechanisms have been put forward to explain the maintenance of polymorphism: apostatic, disruptive and sexual selection. All of them make partly different predictions. Our aims were to investigate evolutionary causes and adaptive functions of colour polymorphism by taking into account a number of ecological and morphological features of polymorphic species. Overall, we found 334 species showing colour polymorphism, which is 3.5% of all bird species. The occurrence of colour polymorphism was very high in Strigiformes, Ciconiiformes, Cuculiformes and Galliformes. Phylogenetically corrected analysis using independent contrasts revealed that colour polymorphism was maximally expressed in species showing a daily activity rhythm extended to day/night, living in both open and closed habitats. All these findings support the hypothesis that colour polymorphism probably evolved under selective pressures linked to bird detectability as affected by variable light conditions during activity period. Thus, we conclude that selective agents may be prey, predators and competitors, and that colour polymorphism in birds may be maintained by disruptive selection.
Species richness in agamid lizards: chance, body size, sexual selection or ecology?
Authors: Stuart-Fox Devi , Owens Ian P. F.
Published: June 2003

Why does species richness vary so greatly across lineages? Traditionally, variation in species richness has been attributed to deterministic processes, although it is equally plausible that it may result from purely stochastic processes. We show that, based on the best available phylogenetic hypothesis, the pattern of cladogenesis among agamid lizards is not consistent with a random model, with some lineages having more species, and others fewer species, than expected by chance. We then use phylogenetic comparative methods to test six types of deterministic explanation for variation in species richness: body size, life history, sexual selection, ecological generalism, range size and latitude. Of eight variables we tested, only sexual size dimorphism and sexual dichromatism predicted species richness. Increases in species richness are associated with increases in sexual dichromatism but reductions in sexual size dimorphism. Consistent with recent comparative studies, we find no evidence that species richness is associated with small body size or high fecundity. Equally, we find no evidence that species richness covaries with ecological generalism, latitude or range size.
Caste fate conflict in swarm-founding social Hymenoptera: an inclusive fitness analysis
Authors: Wenseleers T. , Ratnieks F. L. W. , Billen J.
Published: June 2003

A caste system in which females develop into morphologically distinct queens or workers has evolved independently in ants, wasps and bees. Although such reproductive division of labour may benefit the colony it is also a source of conflict because individual immature females can benefit from developing into a queen in order to gain greater direct reproduction. Here we present a formal inclusive fitness analysis of caste fate conflict appropriate for swarm-founding social Hymenoptera. Three major conclusions are reached: (1) when caste is self-determined, many females should selfishly choose to become queens and the resulting depletion of the workforce can substantially reduce colony productivity; (2) greater relatedness among colony members reduces this excess queen production; (3) if workers can prevent excess queen production at low cost by controlled feeding, a transition to nutritional caste determination should occur. These predictions generalize results derived earlier using an allele-frequency model [Behav. Ecol. Sociobiol. (2001) 50: 467] and are supported by observed levels of queen production in various taxa, especially stingless bees, where caste can be either individually or nutritionally controlled.
Sex-specific reaction norms to intraspecific larval competition in the mosquito Aedes aegypti
Authors: Bedhomme S. , Agnew P. , Sidobre C. , Michalakis Y.
Published: June 2003

As the relationship between a given life-history trait and fitness is not necessarily the same for the two sexes, an ‘intersexual ontogenetic conflict’ may arise. We analysed the phenotypic reaction to intraspecific larval competition of the mosquito, Aedes aegypti, asking: (i) Do both sexes pay the cost of competition with the same life-history traits and are they equal competitors? (ii) Is there a specific cost of competition beyond sharing food resources? We found that competition incurs a specific cost that was expressed differently by the two sexes. Indeed, each sex maintained the more important life-history trait(s) for their fitness (developmental time for males and body weight and size for females) at the expense of other traits, thus minimizing the effects of competition on their fitness. The competition exerted by females was estimated as being more intense, probably linked with the greater importance of body size for their fitness.
Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms
Authors: Borsch T. , Hilu K. W. , Quandt D. , Wilde V. , Neinhuis C. , Barthlott W.
Published: June 2003

Recent contributions from DNA sequences have revolutionized our concept of systematic relationships in angiosperms. However, parts of the angiosperm tree remain unclear. Previous studies have been based on coding or rDNA regions of relatively conserved genes. A phylogeny for basal angiosperms based on noncoding, fast-evolving sequences of the chloroplast genome region trnT-trnF is presented. The recognition of simple direct repeats allowed a robust alignment. Mutational hot spots appear to be confined to certain sectors, as in two stem-loop regions of the trnL intron secondary structure. Our highly resolved and well-supported phylogeny depicts the New Caledonian Amborella as the sister to all other angiosperms, followed by Nymphaeaceae and an Austrobaileya–Illicium–Schisandra clade. Ceratophyllum is substantiated as a close relative of monocots, as is a monophyletic eumagnoliid clade consisting of Piperales plus Winterales sister to Laurales plus Magnoliales. Possible reasons for the striking congruence between the trnT-trnF based phylogeny and phylogenies generated from combined multi-gene, multi-genome data are discussed.
Sequences and structural organization of phospholipase A2 genes from Vipera aspis aspis, V. aspis zinnikeri and Vipera berus berus venom
Authors: Guillemin Isabelle , Bouchier Christiane , Garrigues Thomas , Wisner Anne , Choumet Valérie
Published: June 2003

We used a PCR‐based method to determine the genomic DNA sequences encoding phospholipases A2 (PLA2s) from the venoms of Vipera aspis aspis (V. a. aspis), Vipera aspis zinnikeri (V. a. zinnikeri), Vipera berus berus (V. b. berus) and a neurotoxic V. a. aspis snake (neurotoxic V. a. aspis) from a population responsible for unusual neurotoxic envenomations in south‐east France. We sequenced five groups of genes, each corresponding to a different PLA2. The genes encoding the A and B chains of vaspin from the neurotoxic V. a. aspis, PLA2‐I from V. a. zinnikeri, and the anticoagulant PLA2 from V. b. berus are described here. Single nucleotide differences leading to amino‐acid substitutions were observed both between genes encoding the same PLA2 and between genes encoding different PLA2s. These differences were clustered in exons 3 and 5, potentially altering the biological activities of PLA2. The distribution and characteristics of the PLA2 genes differed according to the species or subspecies. We characterized for the first time genes encoding neurotoxins from the V. a. aspis and V. b. berus snakes of central France. Genes encoding ammodytins I1 and I2, described previously in Vipera ammodytes ammodytes (V. am. ammodytes), were also present in V. a. aspis and V. b. berus. Three different ammodytin I1 gene sequences were characterized: one from V. b. berus, the second from V. a. aspis, V. a. zinnikeri and the neurotoxic V. a. aspis, and the third from the neurotoxic V. a. aspis. This third sequence was identical with the reported sequence of the V. am. ammodytes ammodytin I1 gene. Genes encoding monomeric neurotoxins of V. am. ammodytes venom, ammodytoxins A, B and C, and the Bov‐B LINE retroposon, a phylogenetic marker found in V. am. ammodytes genome, were identified in the genome of the neurotoxic V. a. aspis. These results suggest that the population of neurotoxic V. a. aspis snakes from south‐east France may have resulted from interbreeding between V. a. aspis and V. am. ammodytes.
1H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2‐avid hemoglobin from the trematode Paramphistomum epiclitum
Authors: Du Weihong , Xia Zhicheng , Dewilde Sylvia , Moens Luc , La Mar Gerd N.
Published: June 2003

The solution molecular and electronic structures of the active site in the extremely O2‐avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1H NMR on the cyanomet form in order to elucidate the distal hydrogen‐bonding to a ligated H‐bond acceptor ligand. Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild‐type Hb more closely resembles the crystal structure of the recombinant wild‐type than the true wild‐type met‐hemoglobin. The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures. Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H‐bond to the bound cyanide. The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide. The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted ≈ 10° from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Oη. The pattern of heme contact shifts with large low‐field shifts for 7‐CH3 and 18‐CH3 is shown to arise not from the 180° rotation about the α‐γ‐meso axis, but due to the ≈ 45° rotation of the axial His imidazole ring, relative to that in mammalian globins.
Etoposide upregulates Bax‐enhancing tumour necrosis factor‐related apoptosis inducing ligand‐mediated apoptosis in the human hepatocellular carcinoma cell line QGY‐7703
Authors: Miao Lin , Yi Peng , Wang Yi , Wu Mian
Published: June 2003

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has attracted much attention because of its ability to kill tumour cells. In this study, we demonstrated that treatment of QGY‐7703 cells with the combination of TRAIL and etoposide resulted in synergistic cytotoxic effects. In dissecting the mechanism underlying this synergistic effect, we found that treatment with etoposide alone resulted in the upregulation of Bax, while the level of truncated Bid (tBid) was unchanged. In contrast, while treatment with TRAIL alone significantly increased the level of tBid, the expression of Bax remained unaffected. The enhanced apoptosis was accompanied by an increased release of cytochrome c and second mitochondria‐derived activator of caspase/direct IAP binding protein with low pI (DIABLO) from mitochondria, leading to the activation of cellular caspase‐8, ‐9, ‐3 and ‐7, as well as poly ADP‐ribose polymerase. This enhanced release of cytochrome c and second mitochondria‐derived activator of caspase/DIABLO was inhibited by the general caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone. The RT–PCR and Western blotting results demonstrated that the levels of both mRNA and protein for death receptor‐4, death receptor‐5 and decoy receptor‐2 remained unchanged in response to etoposide, indicating that the synergistic effect of TRAIL and etoposide is not a result of increasing the expression for TRAIL receptors, but rather is associated with amplification of the mitochondrial signal pathway.
Structural and serological studies on a new 4‐deoxy‐d‐arabino‐hexose‐containing O‐specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6)
Authors: Katzenellenbogen Ewa , Kocharova Nina A. , Zatonsky George V. , Witkowska Danuta , Bogulska Maria , Shashkov Alexander S. , Gamian Andrzej , Knirel Yuriy A.
Published: June 2003

The O‐specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain d‐fucose, l‐rhamnose, 4‐deoxy‐d‐arabino‐hexose and O‐acetyl groups in molar ratios 2 : 1 : 1 : 1. On the basis of methylation analysis and 1H and 13C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O‐specific polysaccharide was established. Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4‐deoxy‐d‐arabino‐hexose‐containing LPS from Citrobacter PCM 1487 (serogroup O5) or C. youngae PCM 1488 (serogroup O36). Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologically distinct from strain PCM 1531 and should be reclassified into another serogroup.
Crystal structure at 3 Å of mistletoe lectin I, a dimeric type‐II ribosome‐inactivating protein, complexed with galactose
Authors: Niwa Hideaki , Tonevitsky Alexander G. , Agapov Igor I. , Saward Steve , Pfüller Uwe , Palmer Rex A.
Published: June 2003

The X‐ray structure of mistletoe lectin I (MLI), a type‐II ribosome‐inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 Å resolution to an R‐factor of 19.9% using 21 899 reflections, with Rfree 24.0%. MLI forms a homodimer (A–B)2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N‐terminal domains of two B‐chains involving weak polar and non‐polar interactions. Consequently, the overall arrangement of sugar‐binding sites in MLI differs from those in monomeric type‐II RIPs: two N‐terminal sugar‐binding sites are 15 Å apart on one side of the dimer, and two C‐terminal sugar‐binding sites are 87 Å apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3‐OH and 4‐OH and hydrophobic contact by an aromatic ring. In addition, at the N‐terminal site 2‐OH forms hydrogen bonds with Asp27 and Lys41, and at the C‐terminal site 3‐OH and 6‐OH undergo water‐mediated interactions and C5 has a hydrophobic contact. MLI is a galactose‐specific lectin and shows little affinity for N‐acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar‐binding sites, and fine sugar specificities of their B‐chains, which could have diverged through evolution from a two‐domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.
The inhibition of Ras farnesylation leads to an increase in p27Kip1 and G1 cell cycle arrest
Authors: Reuveni Hadas , Klein Shoshana , Levitzki Alexander
Published: June 2003

HR12 is a novel farnesyltransferase inhibitor (FTI). We have shown previously that HR12 induces phenotypic reversion of H‐rasV12‐transformed Rat1 (Rat1/ras) fibroblasts. This reversion was characterized by formation of cell–cell contacts, focal adhesions and stress fibers. Here we show that HR12 inhibits anchorage independent and dependent growth of Rat1/ras cells. HR12 also suppresses motility and proliferation of Rat1/ras cells, in a wound healing assay. Rat1 fibroblasts transformed with myristoylated H‐rasV12 (Rat1/myr‐ras) were resistant to HR12. Thus, the effects of HR12 are due to the inhibition of farnesylation of Ras. Cell growth of Rat1/ras cells was arrested at the G1 phase of the cell cycle. Analysis of cell cycle components showed that HR12 treatment of Rat1/ras cells led to elevated cellular levels of the cyclin‐dependent kinase inhibitor p27Kip1 and inhibition of the kinase activity of the cyclin E/Cdk2 complex. This is the first time an FTI has been shown to lead to a rise in p27Kip1 levels in ras‐transformed cells. The data suggest a new mechanism for FTI action, whereby in ras‐transformed cells, the FTI causes an increase in p27Kip1 levels, which in turn inhibit cyclin E/Cdk2 activity, leading to G1 arrest.
Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis
Authors: Aiston Susan , Coghlan Matthew P. , Agius Loranne
Published: June 2003

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase‐3 (GSK‐3) and an allosteric inhibitor of phosphorylase (CP‐91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK‐3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes.

GSK‐3 inhibitors (SB‐216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK‐3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP‐91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.
Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide
Authors: Schmid Bernhard , Finnen Michael J. , Harwood John L. , Jackson Simon K.
Published: June 2003

Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor‐α (TNF‐α) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram‐negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon‐γ (IFN‐γ), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF‐α and interleukin‐6 (IL‐6) was approximately five times greater in cells primed with IFN‐γ than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7‐(3,4,5‐triphenyl‐2‐oxo2,3‐dihydro‐imidazole‐1‐yl)heptane phosphonate) and YM 50201 (3‐hydroxyethyl 5,3′‐thiophenyl pyridine) strongly inhibited (up to 90%) TNF‐α and IL‐6 production in response to LPS in both unprimed MonoMac‐6 cells and in cells primed with IFN‐γ. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence‐based amplification methods showed that SK&F 98625 inhibited TNF‐α production by decreasing TNF‐α mRNA levels in MonoMac‐6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.
Selection of peptides inhibiting a β‐lactamase‐like activity
Authors: Yribarren Anne‐Sophie , Thomas Daniel , Friboulet Alain , Avalle Bérangère
Published: June 2003

A library of random peptide sequences was used to select peptides that inhibit an anti‐idiotypic catalytic Ig, immunoglobulin (IgG) 9G4H9, with a β‐lactamase‐like activity. This library displays cyclic heptapeptides on the surface of bacteriophages and represents a collection of up to 4.5 × 109 peptides. The first selection step aimed at enriching the library in species that bind to the whole Ig molecule. The second step was to discriminate peptides that bind to part of the molecule other than the active site. Selected peptides were then screened by surface plasmon resonance analysis. Those displaying measurable Kd values were assayed for their ability to inhibit the catalytic Ig.
Mechanism of protection of peroxidase activity by oscillatory dynamics
Authors: Olsen Lars F. , Hauser Marcus J. B. , Kummer Ursula
Published: June 2003

The peroxidase–oxidase reaction is known to involve reactive oxygen species as intermediates. These intermediates inactivate many types of biomolecules, including peroxidase itself. Previously, we have shown that oscillatory dynamics in the peroxidase–oxidase reaction seem to protect the enzyme from inactivation. It was suggested that this is due to a lower average concentration of reactive oxygen species in the oscillatory state compared to the steady state. Here, we studied the peroxidase–oxidase reaction with either 4‐hydroxybenzoic acid or melatonin as cofactors. We show that the protective effect of oscillatory dynamics is present in both cases. We also found that the enzyme degradation depends on the concentration of the cofactor and on the pH of the reaction mixture. We simulated the oscillatory behaviour, including the oscillation/steady state bistability observed experimentally, using a detailed reaction scheme. The computational results confirm the hypothesis that protection is due to lower average concentrations of superoxide radical during oscillations. They also show that the shape of the oscillations changes with increasing cofactor concentration resulting in a further decrease in the average concentration of radicals. We therefore hypothesize that the protective effect of oscillatory dynamics is a general effect in this system.
Expression of two [Fe]‐hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions
Authors: Forestier Marc , King Paul , Zhang Liping , Posewitz Matthew , Schwarzer Sarah , Happe Thomas , Ghirardi Maria L. , Seibert Michael
Published: June 2003

We have isolated and characterized a second [Fe]‐hydrogenase gene from the green alga, Chlamydomonas reinhardtii. The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [Fe]‐hydrogenases. We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology‐based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2‐gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N‐terminal Fe‐S centers and the existence of extra sequences in the algal enzymes. To our knowledge, this work represents the first systematic study of expression of two algal [Fe]‐hydrogenases in the same organism.
Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity
Authors: Romestand Bernard , Molina Franck , Richard Véronique , Roch Philippe , Granier and Claude
Published: June 2003

To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram‐positive and Gram‐negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25–33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non‐naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the α‐helical part or to the β‐strands of MGD1 had no detectable activity. The bacteriostatic activity of the sequence 25–33 was strictly dependent on the bridging of Cys25 and Cys33 and was proportional to the theoretical isoelectric point of the peptide, as deduced from the variation of activity in a set of peptide analogues of the 25–33 sequence with different numbers of cationic charges. By using confocal fluorescence microscopy, we found that the cyclic peptides bound to Gram‐positive bacteria without apparent lysis. However, by using a fluorescent dye, we observed that dead bacteria had been permeated by the cyclic peptide 25–33. Sequence comparisons in the family of arthopod defensins indicate that MGD1 belongs to a subfamily of the insect defensins, characterized by the constant occurrence of both positively charged and hydrophobic amino acids in the loop. Modelling studies showed that in the MGD1 structure, positively charged and hydrophobic residues are organized in two layered clusters, which might have a functional significance in the docking of MGD1 to the bacterial membrane.
Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites
Authors: Islam M. Khyrul , Miyoshi Takeharu , Kasuga‐Aoki Harue , Isobe Takashi , Arakawa Takeshi , Matsumoto Yasunobu , Tsuji Naotoshi
Published: June 2003

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho‐phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli‐expressed recombinant enzyme has a specific activity of 937 µmol Pi·min−1·mg−1 protein corresponding to a kcat value of 638 s−1 at 55 °C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose‐dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble‐ and membrane‐bound H+‐PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.
Specific targeting of a DNA‐alkylating reagent to mitochondria
Authors: James Andrew M. , Blaikie Frances H. , Smith Robin A. J. , Lightowlers Robert N. , Smith Paul M. , Murphy Michael P.
Published: June 2003

The selective manipulation of the expression and replication of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria‐targeted DNA‐alkylating reagent. The active alkylating moiety [(11aS)‐8‐hydroxy‐7‐methoxy‐1,2,3,11a‐tetrahydro‐5H‐pyrrolo[2,1‐c][1,4]benzodiazepin‐5‐one (DC‐81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA triplets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC‐81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC‐81 by mitochondria, driven by the large membrane potential across the inner membrane. This compound efficiently alkylated isolated supercoiled, relaxed‐circular or linear plasmid DNA and isolated mtDNA. However mitoDC‐81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100‐fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alkylating reagent. This inability to alkylate mtDNAin situhas significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general.
Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance
Authors: Sigala Barbara , Tsaneva Irina R.
Published: June 2003

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the α‐helices that form this bundle show homology to a putative DNA‐binding motif known as SAP. To investigate the biochemical function of the triple‐helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino‐acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple‐helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA‐binding role of the triple‐helix motif [Ceschini et al. (2001) EMBO J. 20, 6601–6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild‐type and several Ydc2 mutants expressed in Δydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full‐length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA ‘phenotype’. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.
Covalent binding to glutathione of the DNA‐alkylating antitumor agent, S23906‐1
Authors: David‐Cordonnier Marie‐Hélène , Laine William , Joubert Alexandra , Tardy Christelle , Goossens Jean‐François , Kouach Mostafa , Briand Gilbert , Thi Mai Huong Doan , Michel Sylvie , Tillequin Francois , Koch Michel , Leonce Stéphane , Pierre Alain , Bailly Christian
Published: June 2003

The benzoacronycine derivative, S23906‐1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906‐1 was evidenced by EI‐MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S‐blocked derivatives failed to react covalently with S23906‐1. Gel shift assays and fluorescence measurements indicated that the binding of S23906‐1 to DNA and to GSH are mutually exclusive. Binding of S23906‐1 to an excess of GSH prevents DNA alkylation. Additional EI‐MS measurements performed with the mixed diester, S28053‐1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906‐1 towards KB‐3‐1 epidermoid carcinoma cells. The GSH‐induced threefold reduction of the cytotoxicity of S23906‐1 is attributed to the reduced formation of lethal drug–DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug–DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906‐1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906‐1.