When a clonal genome finds its way back to a sexual species: evidence from ongoing but rare introgression in the hybridogenetic water frog complex.

Besides several exceptions, asexual metazoans are usually viewed as ephemeral sinks for genomes, which become 'frozen' in clonal lineages after their emergence from ancestral sexual species. Here, we investigated whether and at what rate the asexuals are able to introgress their genomes back into the parental sexual population, thus more or less importantly affecting the gene pools of sexual species. We focused on hybridogenetic hybrids of western Palaearctic water frogs (Pelophylax esculentus), which originate through hybridization between P. ridibundus and P. lessonae, but transmit only clonal ridibundus genome into their gametes. Although usually mating with P. lessonae, P. esculentus may upon mating with P. ridibundus or another hybrid produce sexually reproducing P. ridibundus offspring with the introgressed ex-clonal genome. We compared the rate of nuclear amplified fragment length polymorphism (AFLP) and mitochondrial introgression in two types of populations, that is, those where P. ridibundus occurs in isolation and those where it lives with the hybridogens. Although significant differentiation (Φpt) between sexual and clonal ridibundus genomes suggested limited gene flow between sexuals and hybridogens, a non-negligible (~5%) proportion of P. ridibundus bore introgressed mtDNA and AFLP markers. Whereas transfer of mtDNA was exclusively unidirectional, introgression of nuclear markers was bidirectional. The proportion of introgressed P. ridibundus was highest in syntopic populations with P. esculentus, proving an ongoing and site-specific interspecific genetic transfer mediated by hybridogenetic hybrids. It turns out that asexual hybrids are not just a sink for genes of sexual species, but may significantly influence the genetic architecture of their sexual counterparts.

Rise and persistence of animal polyploidy: evolutionary constraints and potential.

The past decade has witnessed a tremendous increase in interest in polyploidy, which may partly be related to the development of new powerful genetic and genomic tools. These have provided numerous insights into mainly genetic and genomic consequences of polyploidy, dramatically improving our understanding of the dynamics of the polyploidization process and its importance as a mechanism in animal evolution. In contrast, several other aspects of polyploidization, such as physiology, ecology and development, have received considerably less attention. Our aim is not to make an exhaustive review of current knowledge about animal polyploidy, but rather to thoroughly elaborate on some very fundamental questions which still remain open or even neglected. In particular, we show that properties of new polyploid lineages largely depend upon the proximate way in which they arose, but the evolutionary pathways to polyploidy are often unresolved. To help researchers orientate amongst the number of pathways to polyploidy, we provide an extensive review of particular scenarios proposed in distinct animal taxa. We discuss how polyploidy relates to hybridization, particularly with respect to asexuality, and elaborate on whether clonal triploids may help to overcome the constraints of aneuploidy, thereby serving as a triploid bridge towards the establishment of new polyploid species. We further show that in most animal asexual complexes clonal lineages may become established only under one ploidy level (usually either di- or triploidy), and that it is rather rare to see the coexistence of successful clones of different ploidies. We discuss why the rate of polyploidization is higher in some taxa than in others, and what tools we have to evaluate the rate of polyploidization. Finally, we review some of the immediate physiological and developmental effects of polyploidy which are related to the genome size/cell size relation and show how studies of polyploidy may enhance the study of macroecology and developmental biology. See also the sister article focusing on plants by Weiss-Schneeweiss et al. in this themed issue.

Phylogeny, genetic variability and colour polymorphism of an emerging animal model: the short-lived annual Nothobranchius fishes from southern Mozambique.

Nothobranchius are a group of small, extremely short-lived killifishes living in temporary savannah pools in Eastern Africa and that survive annual desiccation of their habitat as dormant eggs encased in dry mud. One mitochondrial (COI) and three nuclear (CX32.2, GHITM, PNP) loci were used to investigate the phylogenetic relationship of Nothobranchius species from southern and central Mozambique. This group shows marked variation in captive lifespan at both the inter- and intraspecific levels; lifespan varies from a few months to over a year. As their distribution encompasses a steep gradient between semi-arid and humid habitats, resulting in contrasting selection pressures on evolution of lifespan and associated life history traits, Mozambican Nothobranchius spp. have recently become a model group in studies of ageing, age-related disorders and life history evolution. Consequently, intraspecific genetic variation and male colour morph distribution was also examined in the recovered clades. Using Bayesian species tree reconstruction and single loci analyses, three large clades were apparent and their phylogenetic substructure was revealed at the inter- and intra-specific levels within those clades. The Nothobranchius furzeri and Nothobranchius orthonotus clades were strongly geographically structured. Further, it was demonstrated that male colour has no phylogenetic signal in N. furzeri, where colour morphs are sympatric, but is associated with two reciprocally monophyletic groups in Nothobranchius rachovii clade, where colour morphs are parapatric. Finally, our analysis showed that a polymorphism in the Melanocortin1 receptor gene (which controls pigmentation in many vertebrates and was a candidate gene of male colouration in N. furzeri) is unrelated to colour phenotypes of the study species. Our results raise significant implications for future comparative studies of the species and populations analysed in the present work.

Sperm-dependent parthenogens delay the spatial expansion of their sexual hosts.

It has been known for long time that asexual organisms may affect the distribution of sexual taxa. In fact, such phenomenon is inherent in the concept of geographical parthenogenesis. On the other hand, it was generally hypothesized that sperm-dependent asexuals may not exercise the same effect on related sexual population, due to their dependence upon them as sperm-donors. Recently, however, it became clear that sperm-dependent asexuals may directly or indirectly affect the distribution of their sperm-hosts, but rather in a small scale. No study addressed the large-scale biogeographic effect of the coexistence of such asexuals with the sexual species. In our study we were interested in the effect of sexual-asexual coexistence on the speed of spatial expansion of the whole complex. We expand previously published Lotka-Volterra model of the coexistence of sexual and gynogenetic forms of spined loach (Cobitis; Teleostei) hybrid complex by diffusion. We show that presence of sperm-dependent parthenogens is likely to negatively affect the spatial expansion of sexuals, and hence the whole complex, compared to pure sexual population. Given that most of the known sperm-dependent asexual complexes are distributed in areas prone to climate-induced colonization/extinction events, we conclude that such mechanism may be an important agent in determining the biogeography of sexual taxa and therefore requires further attention including empirical tests.

Development of nine polymorphic microsatellite loci in the spined loach, Cobitis taenia, and cross-species amplification in the related species C. elongatoides, C. taurica and C. tanaitica.

Nine polymorphic microsatellite loci were developed for the spined loach, Cobitis taenia (Teleostei: Cobitidae). The loci were validated using 50 individuals from a population in Belgium. Moderate to high levels of polymorphism were detected (two to 11 alleles). In addition, most markers amplified successfully in three closely related taxa that are known to hybridize with C. taenia: C. elongatoides, C. taurica and C. tanaitica. Some of the loci are most likely diagnostic among species. These markers will be valuable for the study of the historical and contemporary interactions within C. taenia and the Cobitis species complex.

Ice age cloning--comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation.

Recent advances in population history reconstruction offered a powerful tool for comparisons of the abilities of sexual and clonal forms to respond to Quaternary climatic oscillations, ultimately leading to inferences about the advantages and disadvantages of a given mode of reproduction. We reconstructed the Quaternary historical biogeography of the sexual parental species and clonal hybrid lineages within the Europe-wide hybrid complex of Cobitis spiny loaches. Cobitis elongatoides and Cobitis taenia recolonizing Europe from separated refuges met in central Europe and the Pontic region giving rise to hybrid lineages during the Holocene. Cobitis elongatoides due to its long-term reproductive contact with the remaining parental species of the complex--C. tanaitica and C. spec.--gave rise to two clonal hybrid lineages probably during the last interglacial or even earlier, which survived the Würmian glaciation with C. elongatoides. These lineages followed C. elongatoides postglacial expansion and probably decreased its dispersal rate. Our data indicate the frequent origins of asexuality irrespective of the parental populations involved and the comparable dispersal potential of diploid and triploid lineages.

Regional long-term co-operation in the field of nuclear and radiation emergency preparedness.

Emergency preparedness is generally covered by methodical and coordinative activities of the International Atomic Energy Agency (IAEA) in Member States of the IAEA and by the European Commission (EC) in EU Member and EU Accession Countries. However, the regional harmonisation of emergency arrangements is an important trend of emergency preparedness. The present paper gives a couple of illustrative examples for a regional co-operation in the field of emergency preparedness in Central Europe and an overview on international exercises in this region. The penultimate section contains an outlook on future activities regarding regional co-operation in Central Europe. The following topics have been suggested inter alia: the harmonisation of intervention criteria and countermeasures, co-ordination in the field of information of the public, comprehensive bi lateral and multilateral exercises, exchange of experts between the national nuclear emergency centres and inter-comparison calculations of the computer codes.

Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation.

Reconstruction of the evolutionary history of asexual lineages undermines their suitability as models for the studies of evolutionary consequences of sexual reproduction. Using molecular tools we addressed the origin, age and maternal ancestry of diploid and triploid asexual lineages arisen through the hybridization between spiny loaches Cobitis elongatoides, C. taenia and C. tanaitica. Reconstructions of the phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes, revealed by sequence analyses, suggest that both hybrid complexes (C. elongatoides-taenia and C. elongatoides-tanaitica) contained several asexual lineages of independent origin. Cobitis elongatoides was the exclusive maternal ancestor of all the C. elongatoides-tanaitica hybrids, whereas within the C. elongatoides-taenia complex, hybridization was reciprocal. In both complexes the low haplotype divergences were consistent with a recent origin of asexual lineages. Combined mtDNA and allozyme data suggest that the triploids arose through the incorporation of a haploid sperm genome into unreduced ova produced by diploid hybrids.

A randomized, controlled trial of remacemide for motor fluctuations in Parkinson's disease.

BACKGROUND: Preclinical studies suggest that glutamate antagonists help ameliorate motor fluctuations in patients with PD treated with levodopa. METHODS: In a multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-ranging study, the authors assessed the safety, tolerability, and efficacy of the glutamate receptor blocker remacemide hydrochloride in 279 patients with motor fluctuations treated with levodopa. The primary objective was to assess the short-term tolerability and safety of four dosage levels of remacemide during 7 weeks of treatment. Patients were also monitored with home diaries and the Unified PD Rating Scale (UPDRS) to collect preliminary data on treatment efficacy. RESULTS: Remacemide was well tolerated up to a dosage of 300 mg/d on a twice daily schedule and 600 mg/d on a four times daily schedule. The most common dosage-related adverse events were dizziness and nausea, as observed in previous studies of remacemide. The percent "on" time and motor UPDRS scores showed trends toward improvement in the patients treated with 150 and 300 mg/d remacemide compared with placebo-treated patients, although these improvements were not significant. CONCLUSION: Remacemide is a safe and tolerable adjunct to dopaminergic therapy for patients with PD and motor fluctuations. Although this study had limited power to detect therapeutic effects, the observed improvement is consistent with studies of non-human primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonian signs and symptoms. Additional studies are warranted to confirm these results over an extended period of observation, and to explore the potential neuroprotective effects of remacemide in slowing the progression of PD.

Stereotactic pallidotomy for the treatment of Parkinson's disease. Efficacy and adverse effects at 6 months in 26 patients.

We evaluated the safety and efficacy of microelectrode-guided stereotactic pallidotomy in patients with advanced Parkinson's disease (PD). Using diagnostic criteria and evaluations outlined in the Core Assessment Programme in Transplantation (CAPIT) protocol, we studied unilateral pallidotomy in 26 patients with advanced idiophatic PD, motor fluctuations, and peak dose dyskinesias. All underwent unilateral stereotactic pallidotomy. Assessments conducted in the "practically defined off" and "best on" states at baseline and at 1 and 6 months postoperatively included Unified Parkinson's Disease Rating Scale (UPDRS) parts II, III, and IV and timed motor testing as outlined in CAPIT. Motor UPDRS in the "off" state improved at 1 and 6 months after surgery (p = 0.002, p = 0.008) Likewise, the sum of individual "off" contralateral motor UPDRS items improved (p = 0.0002, p = 0.0005). The duration (p = 0.0001 at 1 and p = 0.001 at 6 months) and severity (p = 0.003 at 1 and p = 0.0005 at 6 months) of dyskinesia improved, but other aspects of the "on" function were unchanged. Serious adverse effects occurred in eight patients and included one fatal deep and three nonfatal frontal lobe hemorrhages with resultant language or behavioral deficits. Nonhemorrhagic complications included one hemiparesis and three frontal lobe syndromes. Pallidotomy improves PD motor disability in the "off" state. Peak dose dyskinesias are reduced, although other aspects of "on" motor function are unchanged. Although morbidity may limit its use, pallidotomy is effective in targeting particular symptoms such as unremitting dyskinesia and severe "off" motor disability in advanced PD.

On the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelates.

The rate constants for reduction of the flavoenzyme, L-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3-7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of alpha-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the alpha-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Parkinsonism and Parkinson's disease.

Parkinsonism is a disabling disorder frequently noted in the elderly. Its prevalence increases with age and has been associated with an increased risk of mortality. Appreciation of certain presenting and characteristic symptoms aids in determining the appropriate cause and treatment strategies.

Proton concentration jumps and generation of transmembrane pH-gradients by photolysis of 4-formyl-6-methoxy-3-nitrophenoxyacetic acid.

Proton concentration gradients across membranes are important for many biological energy transducing processes. The kinetics of proton dependent processes can be studied by pH-jump methods in which protons are photochemically released. In the following we describe the synthesis and the properties of photolabile 4-formyl-6-methoxy-3-nitrophenoxyacetic acid, a 'caged proton'. The synthesis is based on vanillin, which is alkylated with chloroacetic acid to give a carboxylic acid (pK = 2.72). In a second step a nitro group ortho to the formyl group is introduced. Photochemical proton release occurs by a reaction mechanism analogous to the well known photochemical formation of 2-nitrosobenzoic acid from 2-nitrobenzaldehyde. The pK values of the photoproduct are 0.75 and 2.76, respectively, thus allowing the use of the compound in a wide pH-range. The quantum yield is 0.18, lower than in the case of the 2-nitrobenzaldehyde/2-nitrosobenzoic acid system (phi = 0.5). The release of the proton in a flash photolysis experiment occurs within less than 1 microseconds. The spectrum of photolabile compound has absorption maxima at 263 nm and 345 nm, respectively. Its permeability across a lipid bilayer membrane is very low (permeability coefficient Pd approximately equal to 10(-9) cm.s-1 at pH 8) so that transmembrane proton concentration gradients can be generated.

Effects of lipid structure on the kinetics of carrier-mediated ion transport.

The mechanism of alkali-ion transport mediated by valinomycin (or similar macrocyclic carriers) may be studied using artificial planar bilayer membranes. The rate constants of the single transport steps (association and dissociation of the ion-carrier complex, translocation of free and complexed carrier) can be determined from electrical relaxation experiments. The turnover number of valinomycin which may be calculated from the rate constants is found to be 10(4)-10(5) s-1. Carriers of the valnomycin-type offer the possibility of studying the relationship between membrane structure and transport kinetics. Increasing the chain-length of the lipid strongly reduces the translocation rate constants of the free and the loaded carrier, and also (in the case of lecithin membranes) the association rate constant. Increasing the number of double bonds in the fatty-acid residue of the lipid leads to an inrease of the translocation rate constants. These effects are discussed in terms of microviscosity of the membrane. Addition of cholesterol to monoglyceride membranes seems to affect both the microviscosity and the dipolar potential at the membrane-solution interface.

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli.

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.

Ion channels formed by chemical analogs of gramicidin A.

Channel-forming peptides such as gramicidin A offer the opportunity to study the relationship between chemical structure and transport properties of an ion channel. This article summarizes a number of recent experiments with chemical analogs and derivatives of gramicidin A using artificial lipid bilayer membranes. The introduction of negative charges near the channel mouth leads to an increase in the cation transport rate. Hybrid channels consisting of a neutral and a negatively charged or of a positively and a negatively charged half-channel may be formed. The current-voltage characteristic of these hybrid channels exhibits a pronounced asymmetry. Experiments with charged derivatives of gramicidin A have been used in order to distinguish between different structural models of the dimeric channel; these studies strongly support Urry's model of a single-stranded, head-to-head associated helical dimer. In a further set of experiments gramicidin analogs with modified amino acid sequence were studied. It was found that a single substitution (tryptophan replaced by phenylalanine) leads to marked changes in the conductance of the channel. Analogs with a simplified amino acid sequence such as (L-Trp-D-Leu)7-L-Trp or L-Trp-Gly-(L-Trp-D-Leu)6-L-Trp are able to form cation permeable channels with similar properties as gramicidin A.

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.