bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature and moisture dependence of soil H2 uptake measured in the laboratory
Authors: Smith‐Downey Nicole V. , Randerson James T. , Eiler John M.
Published: July 2006

The soil sink of molecular hydrogen is the largest and most uncertain term in the global atmospheric H2 budget. Lack of information about the mechanisms regulating this sink limits our ability to predict how atmospheric H2 may respond to future changes in climate or anthropogenic emissions. Here we present the results from a series of laboratory experiments designed to systematically evaluate and describe the temperature and soil moisture dependence of H2 uptake by soils from boreal forest and desert ecosystems. We observed substantial H2 uptake between −4°C and 0°C, a broad temperature optimum between 20°C and 30°C, a soil moisture optimum at approximately 20% saturation, and inhibition of uptake at both low and high soil moisture. A sigmoidal function described the temperature response of H2 uptake by soils between −15°C and 40°C. Based on our results, we present a framework for a model of the soil H2 sink.
Compartmentalized cAMP signalling: a personal perspective
Authors: Scott J.D.
Published: July 2006

Scaffolding proteins create order out of chaos. Multifunctional binding proteins such as the AKAPs (A-kinase-anchoring proteins) oversee the dynamic organization of signalling events by clustering activator proteins with kinases, phosphatases and phosphodiesterases and directing them toward their downstream effectors. This article will focus on the role of AKAPs in the spatial and temporal control of cAMP signalling events.
Use of single-cell imaging techniques to assess the regulation of cAMP dynamics
Authors: Willoughby D. , Cooper D.M.F.
Published: July 2006

cAMP is a ubiquitous intracellular signalling molecule that can regulate a wide array of cellular processes. The diversity of action of this second messenger owes much to the localized generation, action and hydrolysis of cAMP within discrete subcellular regions. Further signalling specificity can be achieved by the ability of cells to modulate the frequency or incidence of such cAMP signals. Here, we discuss the use of two cAMP biosensors that measure real-time cAMP changes in the single cell, to address the mechanisms underlying the generation of dynamic cAMP signals. The first method monitors sub-plasmalemmal cAMP changes using mutant cyclic nucleotide-gated channels and identifies an AKAP (A-kinase-anchoring protein)–protein kinase A–PDE4 (phosphodiesterase-4) signalling complex that is central to the generation of dynamic cAMP transients in this region of the cell. The second study uses a fluorescence resonance energy transfer-based cAMP probe, based on Epac1 (exchange protein directly activated by cAMP 1), to examine interplay between Ca2+ and cAMP signals. This study demonstrates real-time oscillations in cAMP driven by a Ca2+-stimulated AC (adenylate cyclase) (AC8) and subsequent PDE4 activity. These studies, using two very different single-cell cAMP probes, broaden our understanding of the specific spatiotemporal characteristics of agonist-evoked cAMP signals in a model cell system.
Peptides for disruption of PKA anchoring
Authors: Hundsrucker C. , Rosenthal W. , Klussmann E.
Published: July 2006

Adaptor or scaffolding proteins are at the basis of multiprotein complexes that spatially and temporally co-ordinate the propagation and integration of a broad range of cellular events. One class of scaffolding proteins are AKAPs (A-kinase-anchoring proteins). They sequester PKA (protein kinase A) and other signalling molecules including phosphodiesterases, other protein kinases and protein phosphatases to specific subcellular compartments. AKAP-dependent protein–protein interactions play a role in many physiologically relevant processes. For example, AKAP–PKA interactions are essential for the vasopressin-mediated water re-absorption in renal collecting duct principal cells or β-adrenoceptor-induced increases in cardiac myocyte contractility. Here, we discuss recently developed peptide disruptors of AKAP–PKA interactions. Such peptides are valuable tools to study the relevance of PKA anchoring in cellular processes.
Phosphodiesterase-4 gates the ability of protein kinase A to phosphorylate G-protein receptor kinase-2 and influence its translocation
Authors: Houslay M.D. , Baillie G.S.
Published: July 2006

Challenge of the β2Ar (β2-adrenergic receptor) with isoprenaline in HEK-293β2 cells (human embryonic kidney cells stably overexpressing a FLAG- and green fluorescent protein-tagged β2Ar) results in the PKA (cAMP-dependent protein kinase) phosphorylation of GRK2 (G-protein receptor kinase-2). This response was enhanced when PDE4 (phosphodiesterase-4) activity was attenuated using either rolipram, a PDE4-selective inhibitor, or with siRNA (small interfering RNA) knockdown of both PDE4B and PDE4D. Rolipram also facilitated GRK2 recruitment to the membrane and phosphorylation of the β2Ar by GRK2 in response to isoprenaline challenge of cells. In resting cells, rolipram treatment alone is sufficient to promote PKA phosphorylation of GRK2, with consequential effects on GRK2 translocation and GRK2 phosphorylation of the β2Ar. Similar effects are observed in cardiac myocytes. We propose that PDE4 activity protects GRK2 from inappropriate phosphorylation by PKA in resting cells that might have occurred through fluctuations in basal cAMP levels. Thus PDE4 gates the action of PKA to phosphorylate GRK2.
The molecular machinery for cAMP-dependent immunomodulation in T-cells
Authors: Taskén K. , Stokka A.J.
Published: July 2006

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.
Layers of organization of cAMP microdomains in a simple cell
Authors: Martin A.C.L. , Cooper D.M.F.
Published: July 2006

Based on a variety of single-cell measurements, the notion that cAMP microdomains exist in cells is being increasingly embraced. The cellular and molecular underpinnings of this organization are also steadily being revealed. A dependence of Ca2+-sensitive ACs (adenylate cyclases) in HEK-293 cells (human embryonic kidney cells) on capacitative Ca2+ entry is enforced by their presence in lipid rafts and protein–protein interactions. In these cells, many of the participants in the cAMP cascade, including AC, phosphodiesterase 4, cAMP-dependent protein kinase [PKA (protein kinase A)] and protein phosphatase 2A, are now seen to be involved in higher order assemblies. Moreover, the presence of Na+/H+ exchanger 1 in these domains creates a microclimate, protected against global swings in cellular pH. The Ca2+-stimulatable AC8, which is targeted to these regions, can sequester calmodulin for its own regulatory purposes. These devices are a sampling of the multiple layers of organization that are in place – even in a simple cell – to ensure faithful and economical communication of the cAMP message.
Functional localization of cAMP signalling in cardiac myocytes
Authors: Vandecasteele G. , Rochais F. , Abi-Gerges A. , Fischmeister R.
Published: July 2006

The cAMP pathway is of cardinal importance for heart physiology and pathology. The spatial organization of the various components of the cAMP pathway is thought to allow the segregation of functional responses triggered by the different neuromediators and hormones that use this pathway. PDEs (phosphodiesterases) hydrolyse cAMP (and cGMP) and play a major role in this process by preventing cAMP diffusion to the whole cytosol and inadequate target activation. The development of olfactory cyclic nucleotide-gated channels to directly monitor cAMP beneath the plasma membrane in real time allows us to gain new insights into the molecular mechanisms responsible for cAMP homoeostasis and hormonal specificity in cardiac cells. The present review summarizes the recent results we obtained using this approach in adult rat ventricular myocytes. In particular, the role of PDEs in the maintenance of specific cAMP signals generated by β-adrenergic receptors and other Gs-coupled receptors will be discussed.
Compartmentalized cAMP signalling is important in the regulation of Ca2+ cycling in the heart
Authors: Lygren B. , Taskén K.
Published: July 2006

Co-ordinated myocyte handling of calcium is essential for efficient excitation–contraction coupling in the heart. The calcium cycling activity can be modulated by adrenergic stimulation and subsequent phosphorylation. Important functional consequences of phosphorylation include a greater influx of calcium through the voltage-dependent L-type Ca2+ channel and a greater release of calcium from SR (sarcoplasmic reticulum) through the ryanodine R2 receptor. Furthermore, a more efficient reuptake through SERCA2 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 2) is a result of phosphorylation of its regulatory protein phospholamban. Compartmentalized signalling is important in this signalling cascade, and A-kinase-anchoring proteins play a central role by providing a high level of specificity.
Soluble adenylate cyclase reveals the significance of compartmentalized cAMP on endothelial cell barrier function
Authors: Sayner S. , Stevens T.
Published: July 2006

Within pulmonary microvascular endothelial cells, activation of endogenous adenylate cyclase generates tightly regulated cAMP transitions in the subplasma membrane space. These cAMP fluxes strengthen contacts between adjacent cells to tighten their barrier function. However, pathogenic bacteria have devised a mechanism to transfer toxic adenylate cyclases into eukaryotic cells and generate a cytosolic pool of cAMP that disrupts the barrier. To determine whether membrane or cytosolic cAMP synthesis dominates in control of endothelial cell barrier function, we expressed a soluble mammalian adenylate cyclase chimaera. This chimaera is not constitutively active, but is activated by forskolin. Thus forskolin application increases cAMP in both the plasma membrane and cytosolic compartments. Forskolin induced inter-endothelial cell gaps in cells expressing the soluble adenylate cyclase, demonstrating that the cytosolic cAMP pool dominates over the plasma membrane cAMP pool in control of endothelial cell barrier strength. Indeed, when the soluble chimaera is relocalized to the plasma membrane, the forskolin-stimulated adenylate cyclase activity does not induce gaps. These results therefore support the growing paradigm that membrane and cytosolic cAMP pools target discrete effectors to control different physiological processes.
Restricted diffusion of a freely diffusible second messenger: mechanisms underlying compartmentalized cAMP signalling
Authors: Zaccolo M. , Di Benedetto G. , Lissandron V. , Mancuso L. , Terrin A. , Zamparo I.
Published: July 2006

It is becoming increasingly evident that the freely diffusible second messenger cAMP can transduce specific responses by localized signalling. The machinery that underpins compartmentalized cAMP signalling is only now becoming appreciated. Adenylate cyclases, the enzymes that synthesize cAMP, are localized at discrete parts of the plasma membrane, and phosphodiesterases, the enzymes that degrade cAMP, can be targeted to selected subcellular compartments. A-kinase-anchoring proteins then serve to anchor PKA (protein kinase A) close to specific targets, resulting in selective activation. The specific activation of such individual subsets of PKA requires that cAMP is made available in discrete compartments. In this presentation, the molecular and structural mechanisms responsible for compartmentalized PKA signalling and restricted diffusion of cAMP will be discussed.
cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells
Authors: Dyachok O. , Sågetorp J. , Isakov Y. , Tengholm A.
Published: July 2006

Activation of hormone receptors was recently found to evoke oscillations of the cAMP concentration ([cAMP]) beneath the plasma membrane of insulin-secreting cells. Here we investigate how different time courses of cAMP signals influence the generation of cytoplasmic Ca2+ signals and nuclear translocation of the PKA (protein kinase A) catalytic subunit in individual INS-1 β-cells. [cAMP] was measured with a fluorescent translocation biosensor and ratiometric evanescent wave microscopy. Analysis of PKA nuclear translocation was performed with epifluorescence microscopy and FlAsH (fluorescein arsenical helix binder) labelling of tetracysteine-tagged PKA-Cα subunit. Both oscillatory and stable elevations of [cAMP] induced by intermittent or constant inhibition of phosphodiesterases with isobutylmethylxanthine evoked Ca2+ spiking. During [cAMP] oscillations, the Ca2+ spiking was restricted to the periods of elevated [cAMP]. In contrast, only stable [cAMP] elevation induced nuclear entry of FlAsH-labelled PKA-Cα. These results indicate that oscillations of [cAMP] lead to selective target activation by restricting the spatial redistribution of PKA.
Identification of the macromolecular complex responsible for PI3Kγ-dependent regulation of cAMP levels
Authors: Perino A. , Ghigo A. , Damilano F. , Hirsch E.
Published: July 2006

PI3Kγ is a phosphoinositide 3-kinase characterized by both lipid and protein kinase activity. It is activated by G-protein-coupled receptors and is predominantly expressed in leucocytes; in addition, recent work showed its presence in the heart and its involvement in regulating cardiac functions. In this tissue, PI3Kγ acts as a negative modulator of contractility, by decreasing cAMP concentration through a kinase-independent mechanism. Indeed, whereas PI3Kγ-deficient mice show an abnormal cAMP elevation, cAMP levels in knock-in mouse mutants, expressing a kinase-dead PI3Kγ, are comparable with wild-type controls. PI3Kγ regulates cardiac cAMP homoeostasis by forming a macromolecular complex containing PDE3B (phosphodiesterase 3B). In this complex, PI3Kγ could regulate PDE3B activity through protein kinase A, a PDE activator.
cAMP phosphodiesterase-4A1 (PDE4A1) has provided the paradigm for the intracellular targeting of phosphodiesterases, a process that underpins compartmentalized cAMP signalling
Authors: Huston E. , Houslay T.M. , Baillie G.S. , Houslay M.D.
Published: July 2006

Specificity of cAMP signalling pathways has shown that the intracellular targeting of the individual components confers a three-dimensional context to the signalling paradigms in which they can exquisitely control the specificity of the outcome of the signal. Pivotal to this paradigm is degradation of cAMP by sequestered PDEs (phosphodiesterases). cAMP rapidly diffuses within cells and, without the action of spatially confined PDE populations, cAMP gradients could not be formed and shaped within cells so as to regulate targeted effector proteins. Of particular importance in regulating compartmentalized cAMP signalling are isoforms of the PDE4 family, which are individually defined by unique N-terminal regions. We have developed and pioneered the concept that a major function of this N-terminal region is to confer intracellular targeting of particular PDE4 isoforms on specific signalling complexes and intracellular locations. The paradigm for this concept developed from our original studies on the PDE4A1 (RD1) isoform. The N-terminal region unique to PDE4A1 consists of two well-defined helical regions separated by a mobile hinge region. Helix-2 provides the core membrane-insertion module, with helix-1 facilitating membrane association and fidelity of targeting in living cells. The irreversible, Ca2+-dependent insertion of the N-terminal region of PDE4A1 into membranes provides ‘long-term’ memory of cell activation.
A complex phosphodiesterase system controls β-adrenoceptor signalling in cardiomyocytes
Authors: Mongillo M. , Zaccolo M.
Published: July 2006

β-Adrenergic signalling mediates the positive inotropic effect of catecholamines on cardiomyocytes, mainly through cAMP generation and subsequent activation of PKA (protein kinase A). Given the large diversity of PKA targets within cardiac cells, a precisely regulated and confined activity of such signalling pathways is essential for the specificity of response. PDEs (phosphodiesterases) constitute the only cAMP-degrading mechanism and are expressed in the cardiomyocyte in at least 5 family variants. Each PDE family is characterized by unique functional properties and contributes to a cAMP-degrading system enabling the modulation of PKA activity in a stimulus-dependent fashion.
Signalling from parathyroid hormone
Authors: Tovey S.C. , Dedos S.G. , Taylor C.W.
Published: July 2006

PTH (parathyroid hormone), acting via type 1 PTH receptors, is a major regulator of plasma [Ca2+]. The G-protein, Gs, is an essential component of the sequence linking PTH to plasma Ca2+ regulation, but the relative importance of intracellular signals, including Ca2+ and cAMP, that lie downstream of Gs is not resolved.
Why have sex? The population genetics of sex and recombination
Authors: Otto S.P. , Gerstein A.C.
Published: July 2006

One of the greatest puzzles in evolutionary biology is the high frequency of sexual reproduction and recombination. Given that individuals surviving to reproductive age have genomes that function in their current environment, why should they risk shuffling their genes with those of another individual? Mathematical models are especially important in developing predictions about when sex and recombination can evolve, because it is difficult to intuit the outcome of evolution with several interacting genes. Interestingly, theoretical analyses have shown that it is often quite difficult to identify conditions that favour the evolution of high rates of sex and recombination. For example, fitness interactions among genes (epistasis) can favour sex and recombination but only if such interactions are negative, relatively weak and not highly variable. One reason why an answer to the paradox of sex has been so elusive is that our models have focused unduly on populations that are infinite in size, unstructured and isolated from other species. Yet most verbal theories for sex and recombination consider a finite number of genotypes evolving in a biologically and/or physically complex world. Here, we review various hypotheses for why sex and recombination are so prevalent and discuss theoretical results indicating which of these hypotheses is most promising.
Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation
Authors: Keeney S. , Neale M.J.
Published: July 2006

Homologous recombination is essential for accurate chromosome segregation during meiosis in most sexual organisms. Meiotic recombination is initiated by the formation of DSBs (DNA double-strand breaks) made by the Spo11 protein. We review here recent findings pertaining to protein–protein interactions important for DSB formation, the mechanism of an early step in the processing of Spo11-generated DSBs, and regulation of DSB formation by protein kinases.
The distribution and causes of meiotic recombination in the human genome
Authors: Myers S. , Spencer C.C.A. , Auton A. , Bottolo L. , Freeman C. , Donnelly P. , McVean G.
Published: July 2006

Using the statistical analysis of genetic variation, we have developed a high-resolution genetic map of recombination hotspots and recombination rate variation across the human genome. This map, which has a resolution several orders of magnitude greater than previous studies, identifies over 25000 recombination hotspots and gives new insights into the distribution and determination of recombination. Wavelet-based analysis demonstrates scale-specific influences of base composition, coding context and DNA repeats on recombination rates, though, in contrast with other species, no association with DNase I hypersensitivity. We have also identified specific DNA motifs that are strongly associated with recombination hotspots and whose activity is influenced by local context. Comparative analysis of recombination rates in humans and chimpanzees demonstrates very high rates of evolution of the fine-scale structure of the recombination landscape. In the light of these observations, we suggest possible resolutions of the hotspot paradox.
Meiotic recombination hotspots in plants
Authors: Mézard C.
Published: July 2006

Many studies have demonstrated that the distribution of meiotic crossover events along chromosomes is non-random in plants and other species with sexual reproduction. Large differences in recombination frequencies appear at several scales. On a large scale, regions of high and low rates of crossover have been found to alternate along the chromosomes in all plant species studied. High crossover rates have been reported to be correlated with several chromosome features (e.g. gene density and distance to the centromeres). However, most of these correlations cannot be extended to all plant species. Only a few plant species have been studied on a finer scale. Hotspots of meiotic recombination (i.e. DNA fragments of a few kilobases in length with a higher rate of recombination than the surrounding DNA) have been identified in maize and rice. Most of these hotspots are intragenic. In Arabidopsis thaliana, we have identified several DNA fragments (less than 5 kb in size) with genetic recombination rates at least 5 times higher than the whole-chromosome average [4.6 cM (centimorgan)/Mb], which are therefore probable hotspots for meiotic recombination. Most crossover breakpoints lie in intergenic or non-coding regions. Major efforts should be devoted to characterizing meiotic recombination at the molecular level, which should help to clarify the role of this process in genome evolution.
Crossover promotion and prevention
Authors: Lorenz A. , Whitby M.C.
Published: July 2006

Homologous recombination is an important mechanism for the repair of double-strand breaks in DNA. One possible outcome of such repair is the reciprocal exchange or crossing over of DNA between chromosomes. Crossovers are beneficial during meiosis because, as well as generating genetic diversity, they promote proper chromosome segregation through the establishment of chiasmata. However, crossing over in vegetative cells can potentially result in loss of heterozygosity and chromosome rearrangements, which can be deleterious. Consequently, cells have evolved mechanisms to limit crossing over during vegetative growth while promoting it during meiosis. Here, we provide a brief review of how some of these mechanisms are thought to work.
Control of meiotic recombination in Arabidopsis: role of the MutL and MutS homologues
Authors: Franklin F.C.H. , Higgins J.D. , Sanchez-Moran E. , Armstrong S.J. , Osman K.E. , Jackson N. , Jones G.H.
Published: July 2006

Immunocytochemistry reveals that the Arabidopsis mismatch repair proteins AtMSH4, AtMLH3 and AtMLH1 are expressed during prophase I of meiosis. Expression of AtMSH4 precedes AtMLH3 and AtMLH1 which co-localize as foci during pachytene. Co-localization between AtMSH4 and AtMLH3 occurs, but appears transient. AtMLH3 foci are not detected in an Atmsh4 mutant. However, localization of AtMSH4 is unaffected in Atmlh3, suggesting that recombination may proceed to dHj (double Holliday junction) formation. Mean chiasma frequency in Atmsh4 is reduced to 1.55 compared with 9.86 in wild-type. In contrast with wild-type, the distribution of residual crossovers in Atmsh4 closely fits a Poisson distribution. This is consistent with a two-pathway model for meiotic crossing-over whereby most crossovers occur via an AtMSH4-dependent pathway that is subject to interference, with the remaining crossovers arising via an interference-independent pathway. Loss of AtMLH3 results in an approx. 60% reduction in crossovers. Results suggest that dHj resolution can occur, but in contrast with wild-type where most or all dHjs are directed to form crossovers, the outcome is biased in favour of a non-crossover outcome. The results are compatible with a model whereby the MutL complex maintains or imposes a dHj conformation that ensures crossover formation.
Initiation of homologous chromosome pairing during meiosis
Authors: Jordan P.
Published: July 2006

Following pre-meiotic DNA replication, homologous chromosomes must be paired and become tightly linked to ensure reductional segregation during meiosis I. Therefore initiation of homologous chromosome pairing is vital for meiosis to proceed correctly. A number of factors contribute to the initiation of homologous chromosome pairing including telomere and centromere dynamics, pairing centres, checkpoint proteins and components of the axial element. The present review briefly summarizes recent progress in our understanding of initiation of homologous chromosome pairing during meiosis and discusses the differences that are observed between research organisms.
Factors directing telomere dynamics in synaptic meiosis
Authors: Scherthan H.
Published: July 2006

Meiosis creates haploid cells from diploid progenitors. Homologous chromosomes are moved, paired and segregated from each other in a specialized meiosis I division. A second division that lacks a preceding S-phase produces haploid cells. In prophase I, chromosomes attach with their telomeres to the nuclear envelope and undergo oscillating movements that become restricted to a limited nuclear sector during the widely conserved bouquet stage. Recent observations in budding yeast meiosis suggest that telomere clustering depends on actin, whereas exit from the bouquet stage requires meiotic cohesin. Telomere clustering may also be modulated by progression in recombination. These observations suggest that the unique meiotic nuclear topology and telomere dynamics are regulated at different levels.
Balancing the checks: surveillance of chromosomal exchange during meiosis
Authors: Börner G.V.
Published: July 2006

During meiosis, numerous DSBs (double-strand breaks) are induced along the genome which are processed via several steps into crossovers. Crossovers ensure the faithful segregation of homologous chromosomes during meiosis I. Although required for faithful chromosome segregation, DSBs pose a severe hazard to genome integrity. Chromosome segregation in the presence of persisting DSBs can result in loss or missegregation of entire chromosome arms and in the formation of aneuploid gametes, conditions frequently associated with birth defects, still births and cancer susceptibility in offspring. Co-ordination between chromosomal exchange and meiotic cell-cycle progression is achieved via a surveillance mechanism commonly referred to as the recombination checkpoint. Both components of the mitotic DNA damage checkpoint as well as meiosis-specific functions contribute to this highly conserved surveillance system.
Analysis of a cross between green and red fluorescent trypanosomes
Authors: Gibson W. , Peacock L. , Ferris V. , Williams K. , Bailey M.
Published: July 2006

Trypanosoma brucei undergoes genetic exchange in its insect vector, but the mechanism is unknown and no one has yet seen the process. By crossing genetically engineered red and green fluorescent trypanosomes, we have been able to pinpoint the location of genetic exchange in the fly and search for intermediate stages. In experimental crosses of red and green parental trypanosomes, yellow hybrid trypanosomes first appeared in the fly salivary glands as early as 13 days after infection and were observed only in flies with a mixture of red and green trypanosomes in one or both salivary glands. Despite high numbers of flies with mixed infections, yellow trypanosomes were not detected in the fly midgut or proventriculus. The hybrid nature of yellow trypanosomes was confirmed by analysis of molecular karyotypes and microsatellite alleles. As well as yellow hybrids, hybrid trypanosomes with red, green or no fluorescence were also recovered from fly salivary glands. Analysis of microsatellite alleles in parental and progeny clones showed Mendelian inheritance. Our findings are consistent with the hypothesis that mating takes place between trypanosomes in the salivary glands of the fly before they attach to the salivary gland epithelium.
Effects of intra-gene fitness interactions on the benefit of sexual recombination
Authors: Watson R.A. , Weinreich D.M. , Wakeley J.
Published: July 2006

Whereas spontaneous point mutation operates on nucleotides individually, sexual recombination manipulates the set of nucleotides within an allele as an essentially particulate unit. In principle, these two different scales of variation enable selection to follow fitness gradients in two different spaces: in nucleotide sequence space and allele sequence space respectively. Epistasis for fitness at these two scales, between nucleotides and between genes, may be qualitatively different and may significantly influence the advantage of mutation-based and recombination-based evolutionary trajectories respectively. We examine scenarios where the genetic sequence within a gene strongly influences the fitness effect of a mutation in that gene, whereas epistatic interactions between sites in different genes are weak or absent. We find that, in cases where beneficial alleles of a gene differ from one another at several nucleotide sites, sexual populations can exhibit enormous benefit compared with asexual populations: not only discovering fit genotypes faster than asexual populations, but also discovering high-fitness genotypes that are effectively not evolvable in asexual populations.
Sex-ratio meiotic drive in Drosophila simulans: cellular mechanism, candidate genes and evolution
Authors: Montchamp-Moreau C.
Published: July 2006

The sex-ratio trait, reported in a dozen Drosophila species, is a type of naturally occurring meiotic drive in which the driving elements are located on the X chromosome. Typically, as the result of a shortage of Y-bearing spermatozoa, males carrying a sex-ratio X chromosome produce a large excess of female offspring. The presence of sex-ratio chromosomes in a species can have considerable evolutionary consequences, because they can affect individual fitness and trigger extended intragenomic conflict. Here, I present the main results of the study performed in Drosophila simulans. In this species, the loss of Y-bearing spermatozoa is related to the inability of the Y chromosome sister-chromatids to separate properly during meiosis II. Fine genetic mapping has shown that the primary sex-ratio locus on the X chromosome contains two distorter elements acting synergistically, both of which are required for drive expression. One element has been genetically mapped to a tandem duplication. To infer the natural history of the trait, the pattern of DNA sequence polymorphism in the surrounding chromosomal region is being analysed in natural populations of D. simulans harbouring sex-ratio X chromosomes. Initial results have revealed the recent spread of a distorter allele.
Why Mendelian segregation?
Authors: Úbeda F.
Published: July 2006

The discovery of alleles that are able to distort segregation during meiosis in their favour raises the question of why Mendelian segregation is the rule and segregation distortion the exception. Previous research on this topic was limited by an unrealistic assumption: equal segregation in the two sexes. Úbeda and Haig [(2005) Genetics 170, 1345–1357] provide a new model allowing sex-specific segregation distortion. This model shows that natural selection favours departure from Mendelian expectations. The evolutionary instability of Mendelian segregation under more realistic assumptions requires a new paradigm that explains its ubiquity.
Genetic conflicts during meiosis and the evolutionary origins of centromere complexity
Authors: Malik H.S. , Bayes J.J.
Published: July 2006

Centromeric DNA evolves rapidly, ranging in size and complexity over several orders of magnitude. Traditional attempts at studying centromeres have left unexplained the causes underlying this complexity and rapid evolution. Instead of directly studying centromeric DNA sequence, our approach has been to study the proteins that epigenetically determine centromere identity. We have discovered that centromeric histones (CenH3s) have evolved under positive selection in multiple lineages, suggesting an involvement in recurrent genetic conflict. Our hypothesis is that ‘centromere-drive’ is the source of this conflict. Under this model, centromeres compete via microtubule attachments for preferential transmission in female meioses occurring in animals and plants. Since only one of four meiotic products will become the egg, this competition confers a selfish advantage to chromosomes that can make more microtubule attachments, resulting in runaway expansions of centromeric satellites. While beneficial to the ‘driving’ chromosome, these expansions can have deleterious effects on the fitness of an organism and of the species. CenH3s as well as other heterochromatin proteins have evolved under positive selection to suppress the deleterious consequences of ‘centromere-drive’ by restoring meiotic parity.
Relationship of recombination patterns and maternal age among non-disjoined chromosomes 21
Authors: Sherman S.L. , Lamb N.E. , Feingold E.
Published: July 2006

Advancing maternal age has long been identified as the primary risk factor for human chromosome trisomy. More recently, altered patterns of meiotic recombination have been found to be associated with non-disjunction. We have used trisomy 21 as a model for human non-disjunction that occurs during the formation of oocytes to understand the role of maternal age and recombination. Patterns of recombination that increase the risk for non-disjunction of chromosome 21 include absence of any exchange, an exchange near the centromere or a single, telomeric exchange. Our recent work has shown that different susceptibility patterns are associated with the origin of the meiotic error and maternal age. For MI (meiosis I) errors, the proportion of oocytes with susceptible recombination patterns is highest among young mothers and decreases significantly in the oldest age group. In fact, the pattern of exchanges among the oldest age group mimics the pattern observed among normally disjoining chromosomes 21. These results suggest that oocytes of younger women, with functional meiotic apparatus and/or robust ovarian environment, are able to properly resolve all but the most susceptible exchange patterns. As women age, however, meiotic mechanisms erode, making it difficult to resolve even stable exchange events. Interestingly, our preliminary recombination results on MII errors reveal the opposite relationship with maternal age: susceptible pericentromeric exchanges occur most often in the older age group compared with the younger age group. If confirmed, we will have further evidence for multiple risk factors for non-disjunction that act at different times in the meiotic process.
Intra-allelic mutation at human telomeres
Authors: Britt-Compton B. , Baird D.M.
Published: July 2006

The maintenance of telomere length is important in upholding the integrity of the genome. However, it is clear from detailed observations of both telomere length and internal repeat structure that human telomeres are extremely dynamic structures and are subjected to multiple processes that create considerable heterogeneity. Genetic evidence suggests that meiotic recombination within telomeres is rare. However, there are various lines of evidence that implicate the involvement of intra-allelic processes in human telomere dynamics. In this paper, we briefly review some of this evidence and the putative mechanisms of intra-allelic telomeric mutation.
Differential mitotic checkpoint protein requirements in somatic and germ cells
Authors: Jeganathan K.B. , van Deursen J.M.
Published: July 2006

Cdc20 (cell division cycle 20) and Cdh1 are the activating subunits of APC (anaphase-promoting complex), an E3-ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation due to early degradation of cyclin B and securin, mitotic checkpoint protein complexes consisting of BubR1, Bub3 and Mad2 bind to and inhibit APCCdc20 until all chromosomes are properly attached to the mitotic spindle and aligned in the metaphase plate. The nuclear transport factors Rae1 and Nup98, which convert into mitotic checkpoint proteins in M-phase, further prevent chromosome missegregation by assembling into a complex with APCCdh1 and delaying APCCdh1-mediated ubiquitination of securin. Disruption of Mad2, BubR1, Bub3 or Rae1 in mice results in substantial aneuploidy in somatic tissues, but whether these genes are equally important for accurate chromosome segregation during meiosis has not yet been established. To address this issue, we generated cohorts of male mice in which Mad2, BubR1, Bub3, Rae1 and Nup98 were disrupted either individually or in combination. We tested the fertility of these mice and performed chromosome counts on secondary spermatocytes. We found that male fertility and accurate chromosome segregation during spermatogenesis are highly dependent on BubR1, but not Mad2, Bub3, Rae1 and Nup98. Our results suggest that the mechanisms ensuring accurate chromosome segregation differ between mitotic and meiotic cells.
NGF receptor TrkAd5: therapeutic agent and drug design target
Authors: Dawbarn D. , Fahey M. , Watson J. , Tyler S. , Shoemark D. , Sessions R. , Zhang R. , Brady L. , Willis C. , Allen S.J.
Published: July 2006

Biochemical studies have shown that domain 5 of the TrkA (tropomyosin receptor kinase A) receptor is involved in the binding of NGF (nerve growth factor). Crystallographic studies have confirmed this, demonstrating that one homodimer of NGF binds to two TrkAd5 molecules. TrkAd5 has been made recombinantly in Escherichia coli, purified and shown to bind NGF with picomolar affinity. We have used the co-ordinates of the crystal structure of the NGF–TrkAd5 complex to screen approximately two million compounds in silico for the identification of small molecule agonists/antagonists. Selected hits were shown to be active in an in vitro ligand-binding assay; structure–activity relationships are now being investigated. In addition, TrkAd5 has been shown to be efficacious in preclinical models of inflammatory pain and asthma by the sequestration of excess levels of endogenous NGF, and therefore represents a novel therapeutic agent.
Neuro–immune interaction in allergic asthma: role of neurotrophins
Authors: Nassenstein C. , Kutschker J. , Tumes D. , Braun A.
Published: July 2006

The nature of persistent airway hyperreactivity and chronic inflammation in asthma remains unclear. It has been suggested that bi-directional neuro–immune interaction plays an important role in the pathogenesis of this disease, leading to enhanced airway narrowing after contact with unspecific stimuli, as well as infiltration, activation and degranulation of several immune cell subtypes. Important mediators in neuro-immune cross-talk are neurotrophins, which are produced by cells at the site of inflammation. In addition to modulating the function of several leucocyte subsets, they play an important role in the synthesis of neuropeptides by sensory nerve cells. Neuropeptides have been shown to cause smooth-muscle contraction and, in addition, modulate the production of pro-inflammatory molecules by leucocytes. The aim of the present review is to provide an overview of the molecular mechanisms by which neurotrophins and neuropeptides are involved in neuro–immune cross-talk in allergic asthma.
The contribution of neurotrophins to the pathogenesis of allergic asthma
Authors: Rochlitzer S. , Nassenstein C. , Braun A.
Published: July 2006

The neurotrophins nerve growth factor, brain-derived neurotrophic factor, NT-3 (neurotrophin 3) and NT-4 are known for regulating neuron development, function and survival. Beyond this, neurotrophins were found to exert multiple effects on non-neuronal cells such as immune cells, smooth muscle and epithelial cells. In allergic asthma, airway inflammation, airway obstruction, AHR (airway hyperresponsiveness) and airway remodelling are characteristic features, indicating an intensive interaction between neuronal, structural and immune cells in the lung. In allergic asthma patients, elevated neurotrophin levels in the blood and locally in the lung are commonly observed. Additionally, structural cells of the lung and immune cells, present in the lung during airway inflammation, were shown to be capable of neurotrophin production. A functional relationship between neurotrophins and the main features of asthma was revealed, as airway obstruction, airway inflammation, AHR and airway remodelling were all shown to be stimulated by neurotrophins. The aim of the present review is to provide an overview of neurotrophin sources and target cells in the lung, concerning their possible role as mediators between structural cells, immune cells and neurons, connecting the different features of allergic asthma.
Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain
Authors: Soulé J. , Messaoudi E. , Bramham C.R.
Published: July 2006

Interest in BDNF (brain-derived neurotrophic factor) as an activity-dependent modulator of neuronal structure and function in the adult brain has intensified in recent years. Localization of BDNF and its receptor tyrosine kinase TrkB (tropomyosin receptor kinase B) to glutamate synapses makes this system attractive as a dynamic, activity-dependent regulator of excitatory transmission and synaptic plasticity in the adult brain. Development of stable LTP (long-term potentiation) in response to high-frequency stimulation requires new gene expression and protein synthesis, a process referred to as synaptic consolidation. Several lines of evidence have implicated endogenous BDNF–TrkB signalling in synaptic consolidation. This mini-review emphasizes new insights into the molecular mechanisms underlying this process. The immediate early gene Arc (activity-regulated cytoskeleton-associated protein) is strongly induced and transported to dendritic processes after LTP induction in the dentate gyrus in live rats. Recent work suggests that sustained synthesis of Arc during a surprisingly protracted time-window is required for hyperphosphorylation of actin-depolymerizing factor/cofilin and local expansion of the actin cytoskeleton in vivo. Moreover, this process of Arc-dependent synaptic consolidation is activated in response to brief infusion of BDNF. Microarray expression profiling has also revealed a panel of BDNF-regulated genes that may co-operate with Arc during LTP maintenance. In addition to regulating gene expression, BDNF signalling modulates the fine localization and biochemical activation of the translation machinery. By modulating the spatial and temporal translation of newly induced (Arc) and constitutively expressed mRNA in neuronal dendrites, BDNF may effectively control the window of synaptic consolidation. These findings have implications for mechanisms of memory storage and mood control.
Structural and functional properties of mouse proNGF
Authors: Paoletti F. , Konarev P.V. , Covaceuszach S. , Schwarz E. , Cattaneo A. , Lamba D. , Svergun D.I.
Published: July 2006

The unprocessed pro-form of the NGF (nerve growth factor), proNGF (NGF precursor, without signal peptide), has been suggested to have additional functions distinct from its role as a promoter of protein folding, i.e. apoptosis and/or neurotrophic activity. Aiming to gain insights into the specific molecular interactions that mediate proNGF biological activity and into the structural determinants stabilizing its pro-region, rm-proNGF (recombinant mouse proNGF) was expressed in Escherichia coli, refolded in vitro and characterized by physicochemical methods. X-ray solution scattering measurements (small angle X-ray scattering) revealed that rm-proNGF is dimeric in solution and appears to be anisometric when compared with the compact structure of the NGF dimer. Two structural models, a globular crab-like shape and an elongated rod-like shape, equally fit to the experimental results, pointing to an intrinsically structural disordered pro-region of NGF. The models obtained allowed the interpretation of TrkA (tropomyosin receptor kinase A) binding and activation assays in cell cultures, shedding new light on the key role of proNGF in neuronal survival and neurodegeneration.
Functional mimetics of neurotrophins and their receptors
Authors: Peleshok J. , Saragovi H.U.
Published: July 2006

Neurotrophins regulate cell survival, death, differentiation and growth. Neurotrophins and their receptors have been validated for pathologies including neurodegenerative disorders of the central nervous system and the peripheral nervous system, certain types of cancers, asthma, inflammation and others. Development of neurotrophin-based therapeutics is important due to the limitations of using whole neurotrophins as pharmacological agents. The use of mimicry has proven to be an alternative. Mimetics can be developed through a number of different approaches. To develop receptor-binding agents, we have used anti-receptor antibody mimicry and neurotrophin mimicry. To develop ligand-binding agents, we have used antiligand antibody mimicry and receptor mimicry. High-throughput screening can be incorporated to complement any of these approaches. The end result is small molecule peptidomimetics with properties favourable over proteins. The present review will offer a general overview of these strategies with a few proven examples from our laboratory.
Correlated vH2 and jCO product states from formaldehyde photodissociation: Dynamics of molecular elimination
Authors: Chambreau Steven D. , Lahankar Sridhar A. , Suits Arthur G.
Published: July 2006

A detailed study of the photoinduced molecular elimination pathway of formaldehyde on the ground state surface was carried out using high-resolution dc slice ion imaging. Detailed correlated H2 rovibrational and CO rotational product quantum state distributions were measured by imaging spectroscopically selected CO velocity distributions following photodissociation at energies from ∼1800to∼4100cm−1 above the barrier to molecular elimination. Excitation to the 2141, 2143, 2241, 2243, and 2341 bands of H2CO are reported here. The dependence of the product rovibrational distributions on excitation energy are discussed in light of a dynamical model which has been formulated to describe the strong product state correlations observed.
Vibrational spectra of germanium-carbon clusters in solid Ar: Identification of the ν4(σu) mode of linear GeC5Ge
Authors: Gonzalez E. , Rittby C. M. L. , Graham W. R. M.
Published: July 2006

The linear GeC5Ge cluster has been detected in Fourier transform infrared spectra observed when the products from the dual laser evaporation of carbon and germanium rods were trapped in solid Ar at ∼10K. Comparison of C13 isotopic shift measurements with the predictions of density functional theory calculations at the B3LYP/cc-pVDZ level confirms the identification of the ν4(σu) mode of GeC5Ge at 2158.0cm−1.