Highly efficient synthesis of alternate alpha- and beta-(1-->3)-linked glucose hepta- and octasaccharides

Two heptasaccharides alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-1-OMP and beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp1-OMP, and two octasaccharides alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-1-OMP and beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp1-OMP were synthesized in a stereospecific way by remote control.     

Kinome profiling of sugar signaling in plants using multiple platforms

Although the primary sequence of kinases shows substantial divergence between unrelated eukaryotes, variation in the motifs that are actually phosphorylated by eukaryotic kinases is much smaller. Hence arrays developed for kinome profiling of mammalian cells are useful for kinome profiling of plant tissues as well, facilitating the study of plant signal transduction. We recently employed the Pepscan kinomics chip to reveal the small GTPases in plant sucrose signaling. Here we show that employing a different peptide library (the Pepscan kinase chip) largely similar results are obtained, confirming these earlier data, but such a different library also contributes new insights into the molecular details mediating plant cell responses to a sugar stimulus. Thus when studying plant signal transduction employing peptide arrays, using multiple platforms both increases the confidence of results and provides additional information.Key words: sucrose, arabidopsis, kinome profiling, kinase, MAPK, SnRK, casein kinase, tyrosine phosphorylation, CDC2, AGC kinaseIn our recently published paper1 we analyze the changes in the plant kinome after sucrose feeding compared to control sorbitol feeding of Arabidopsis. We employed kinomics chips (Pepscan Presto, The Netherlands) containing 960 different kinase consensus peptides selected for their importance in mammalian signal transduction. In addition we used kinase chips (Pepscan Presto, The Netherlands), containing 1,152 peptides covering the majority of peptides available through Phosphobase (version 2.0). These chips contain peptides derived from phosphorylation events described in many kingdoms and are taken from animals, plants, fungi and even bacteria.^2,3 Full details as to the peptides spotted can be found at www. pepscanpresto.com. Results of the cluster analysis of three independent biological replicas of kinome profiling after treatment with water, sorbitol, sucrose and glucose are depicted in Figure 3A of our recent paper.¹ A further analysis of this set of experiments was not given, due to a lower than expected biological reproducibility. In contrast to the >0.8 correlation observed for the set described in our recent paper, correlations of below 0.5 were seen for the experimental set analyzed on the kinase1 chips. However, we feel that some observation made based on these kinome profiles contain valuable information, if only to suggest follow-up experiments.Comparison of the kinome profiles of Arabidopsis treated with sucrose or sorbitol for 1 h revealed a set of 93 differentially phosphorylated consensus peptides, with the majority of 59 peptides showing reduced phosphorylation after sucrose treatment (

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

 The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Received: 20 March 1996/Accepted: 1 July 1996     

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

GABA_A receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABA_A receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABA_A receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

Up to 60 different proteins are recruited to the site of clathrin-mediated endocytosis in an ordered sequence. These accessory proteins have roles during all the different stages of clathrin-mediated endocytosis. First, they participate in the initiation of the endocytic event, thereby determining when and where endocytic vesicles are made; later they are involved in the maturation of the clathrin coat, recruitment of specific cargo molecules, bending of the membrane, and finally in scission and uncoating of the nascent vesicle. In addition, many of the accessory components are involved in regulating and coupling the actin cytoskeleton to the endocytic membrane. We will discuss the different accessory components and their various roles. Most of the data comes from studies performed with cultured mammalian cells or yeast cells. The process of endocytosis is well conserved between these different organisms, but there are also many interesting differences that may shed light on the mechanistic principles of endocytosis.Receptor-mediated endocytosis is the process by which eukaryotic cells concentrate and internalize cell surface receptors from the plasma membrane into small (∼50 nm– ∼100 nm diameter) membrane vesicles (Chen et al. 2011; McMahon and Boucrot 2011; Weinberg and Drubin 2012). This mechanism has been studied extensively in mammalian tissue culture cells and in yeast, and despite the evolutionary distance between yeast and mammalian cells the mechanism of receptor-mediated endocytosis in the respective cell types show remarkable similarities. Indeed many of the ∼60 endocytic accessory proteins (EAPs) found in yeast have homologs in mammalian cells, although both cell types also have unique EAPs (McMahon and Boucrot 2011; Weinberg and Drubin 2012).In the following, we briefly describe known yeast and mammalian EAPs (Sigismund et al. 2012; see also Bökel and Brand 2013; Cosker and Segal 2014; Di Fiore and von Zastrow 2014).

Table 1.

Key endocytic proteins in mammals and in yeast

Multi-element fingerprinting and high throughput sequencing identify multiple elements that affect fungal communities in Quercus macrocarpa foliage

Diverse fungal mutualists, pathogens and saprobes colonize plant leaves. These fungi face a complex environment, in which stochastic dispersal interplays with abiotic and biotic filters. However, identification of the specific factors that drive the community assembly seems unattainable. We mined two broad data sets and identified chemical elements, to which dominant molecular operational taxonomic units (OTUs) in the foliage of a native tree respond most extremely. While many associations could be identified, potential complicating issues emerged. Those were related to unevenly distributed OTU frequency data, a large number of potentially explanatory variables and the disproportionate effects of outlier observations.Key words: community assembly, environmental filter, fungi, heavy metal enrichment, nutrient enrichment, oak, Quercus macrocarpaHyperdiverse fungal communities inhabit the foliage of most plants^1,2 and these fungal communities have been reported for virtually every plant that has been examined.³ Baas-Becking hypothesis states that environment selects microbial communities from the abundant and possibly globally distributed propagule pools.⁴ Although the foliage-associated communities—like other microbial communities—are suspected to be sensitive to environmental drivers, determination of the mechanisms that control the assembly of these foliar communities has remained difficult and elusive. Some of the proposed mechanisms include distance limitations to propagule dispersal,^5–7 volume limitations to propagule loads,⁷ or limitations set by the environmental conditions either on the scale of the site of fungal colonization⁸ or more broadly on a landscape level.^6,9 The forces that may control the fungal community assembly are overlaid by additional biotic controls that include compatibilities between the fungi and host species^10,11 or genotypes^6,12 and the competitive or facilitative interactions among the component fungal genotypes.^6,10–13 Although a variety of potential controls for the foliage-associated fungal communities have been speculated, very little consensus exists on the relative importance of the different drivers. For example, while macronutrient and heavy metal enrichment may have an influence on the composition fungal communities¹⁴ and populations,¹⁵ relative importance of various chemical elements in the foliage remains yet to be investigated.To evaluate the use of multi-element fingerprinting data produced by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in combination with high throughput 454-pyrosequencing for determining influential chemical elements in structuring of the leaf-associated fungal communities, we mined a recent dataset¹⁶ that explored the effects of urbanization on the diversity and composition of the fungal communities associated with a native tree Quercus macrocarpa. From a total list of more than 700 non-singleton fungal OTUs, we selected fifty with highest overall frequency to provide an observationrich dataset for elemental effect assessment; these OTUs accounted for 84.5% of all sequences. Even so, many of these OTUs had a number of zero frequencies (Fig. 1), highlighting one of the difficulties in the use of environmental sequencing data. We omitted one OTU (OTU630 with a likely affinity to Trimmatostroma cordae [Mycosphaerellaceae]) that was strongly affected by the original land use design (urbanization; Wilcoxon rank sum test with a Bonferroni adjustment) and therefore unlikely to be representative for the present analyses of elemental drivers. This OTU was replaced with one with the next highest frequency. The frequencies of these 50 OTUs were investigated in the context of concentrations for 29 elements after the omission of five (Ag, Au, C, δ¹³C, δ¹⁵N) in the final analyses because of their strong association with the land use or the difficulty of finding a biological relevance. Of the remaining elements three (Fe, Cr and Ni) had pairwise correlations exceeding 0.98 between the three pairings; others showed no similar high correlations. To allow comparable evaluation across the broad array of elements, all concentrations were standardized to have a mean equal to zero and a standard deviation equal to one.Open in a separate windowFigure 1Rank-ordered distribution of observed frequencies for those OTU s whose frequency had an extreme slope when associated with the concentrations of one or more chemical elements in the mixed effects model. The asterisk denotes one extreme frequency for OTU 313 with a value 0.8636. Numbers in parentheses indicate the number of observations with a frequency equal to zero. The OTU s were assigned to approximate taxa using BLAST:²⁰ 425: Alternaria alternata (Pleosporaceae); 46: Phoma glomerata (Pleosporaceae); 686: Aureobasidium pullulans (Dothioraceae); 520: Davidiella tassiana (Davidiellaceae); 567: Cladosporioum tenuissimum (Davidiellaceae); 313 Oidium heveae (mitosporic Erysiphaceae); 586: Erysiphe hypogena (Erysiphaceae); 671: Mycosphaerella microsora (Mycosphaerellaceae); 555: Pestalotiopsis sp. (Amphisphaeriaceae); 607: Pleiochaeta setosa (incertae sedis).To rank elements according to their magnitude of association with the abundance of each OTU, a total of 1,450 models (50 OTUs times 29 elements) relating element concentration to OTU abundance were fit to the data. For each model, OTU frequency was the dependent variable, element concentration and time (a factor with three levels) were fixed effects, and—to account for the spatial arrangement of the experimental units—random effects associated with tree nested within site were included in the error structure. Time by element interactions were also investigated and tested using a likelihood ratio test. These mixed effect models were fit using R and the package lme4 (www.rproject.org).Statistical “tests of significance” that produce p-values can be sensitive to assumptions or outliers. Because of this and the fact that our analyses evaluated a total of 1,450 models, p-values themselves were not considered a reliable measure of importance when associating elements with OTU frequency. Instead, we emphasized metrics that highlight extraordinary findings rather than rely on tests of statistical significance. This approach facilitates finding few elements that have the strongest effect on OTU frequency. Note that the use of standardized element concentrations (above) provided slope coefficients that are comparable across all models. “Extreme slopes”, i.e., models where the OTU response to element concentration was strongest, were identified as those with estimated slope coefficients in the lower or upper 2.5 percentile, i.e., those farther than 1.8 standard deviations from the mean across all estimated slopes (Fig. 2). Using this approach, we identified a total of 69 models with extreme slopes (Open in a separate windowFigure 2Distribution of estimated slopes (i.e., the slope for element concentration) for a model relating OTU frequency to element concentration, time and a concentration by time interaction, including a tree-nested-within-site random effect. The mean across all 1,450 OTU s is approximately zero; the two vertical lines identify upper and lower 2.5 percentiles, beyond which the slopes were considered extreme (large black symbols). The horizontal line identifies the cut off maximum leverage (0.24), above which the slopes were considered to have observations with high leverage. Models with observations with a high leverage were tested for extreme slopes by refitting without those observations. Models are ranked from bottom to top in order of increasing leverage and the element for which the high-leverage observations and extreme slopes were recorded are identified on the right y-axis.

Table 1

Slopes identified as extreme in our analyses

Human Genetic Disorders of Axon Guidance

This article reviews symptoms and signs of aberrant axon connectivity in humans, and summarizes major human genetic disorders that result, or have been proposed to result, from defective axon guidance. These include corpus callosum agenesis, L1 syndrome, Joubert syndrome and related disorders, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia. Genes mutated in these disorders can encode axon growth cone ligands and receptors, downstream signaling molecules, and axon transport motors, as well as proteins without currently recognized roles in axon guidance. Advances in neuroimaging and genetic techniques have the potential to rapidly expand this field, and it is feasible that axon guidance disorders will soon be recognized as a new and significant category of human neurodevelopmental disorders.The human brain is highly organized and contains a myriad of axon tracts that follow precise pathways and make predictable connections. Model organism research has provided tremendous advances in our understanding of the principles and molecules governing axon growth and guidance. Remarkably, however, only a handful of human disorders resulting from primary errors in these processes have been identified.Traditional tools of the physician have limited sensitivity and specificity to detect human disorders of axon guidance. In particular, congenital synkinesis may be the only physical examination finding that has been attributed to such disorders. Synkinesis is the involuntary and pathological contraction of a muscle simultaneously with contraction of the intended muscle, and is typically reported with hand/finger or eye/eyelid movements and confirmed by electrophysiological studies. Mirror movement synkinesis refers to the contraction of homologous hand/finger muscles bilaterally when one attempts to move only one hand (Schott and Wyke 1981). In humans, 75%–90% of corticospinal tract (CST) fibers normally decussate in the lower medulla. Mirror movement synkinesis occurs in several human disorders with pathological, neuroimaging, and/or electrophysiological evidence of reduced CST decussation, including Joubert, Kallmann, and Klippel-Feil syndromes (Vulliemoz et al. 2005; Cincotta and Ziemann 2008). In some individuals with mirror movements, electrophysiological data are also consistent with bilateral engagement of the motor corticies (Leinsinger et al. 1997). Ocular synkinesis refers to aberrant patterns of eye movement and accompanies various congenital cranial dysinnervation disorders (CCDDs) (Gutowski et al. 2003; Engle 2007), including CFEOM, Duane syndrome, and Marcus Gunn jaw-winking phenomenon (Fig. 1). Finger and ocular movements require precise motor control, and errors in innervation of these muscles may be more easily detected than errors in the wiring of larger muscle groups. If true, this suggests that the clinical exam could fail to recognize many guidance errors in both the peripheral and central nervous system.Open in a separate windowFigure 1.Ocular synkinesis. (A) Child with CFEOM1 and Marcus Gunn jaw-winking phenomenon harboring a KIF21A mutation. His superior branch of the oculomotor nerve is hypoplastic/absent, resulting in bilateral ptosis from lack of appropriate innervation of the levator palpebrae superioris (LPS) muscle, and a downward position of each eye from absent innervation of the superior rectus muscle (left). Marcus Gunn phenomenon (right) is seen as the synkinetic elevation of the left eyelid with a subtle change in jaw position associated with a volitional increase in pterygoid muscle tension. This results from aberrant innervation of the LPS by axons from the motor branch of the trigeminal nerve that also innervates the intended ipsilateral pterygoid muscle. (B) Adult with Duane retraction syndrome harboring a CHN1 mutation. Central gaze reveals mild exotropia (middle). On attempted right gaze (left) and left gaze (right), there is limited horizontal excursion with globe retraction and secondary palpebral fissure narrowing of the adducting eye. Globe retraction results from synkinesis of the medial and lateral recti muscles. (A) Modified with permission from Yamada et al. 2005. Copyright © (2005) American Medial Association. All rights reserved. (B) Modified from Demer et al. 2007. Copyright © (2007) Association for Research in Vision and Ophthalmology. All rights reserved.The physician’s ability to detect disorders of axon guidance has been augmented by classical pathological, radiological, and electrophysiological techniques. Diagnostic radiologic and postmortem neuropathological studies detect overall changes in white matter volume and major abnormalities of axon tracts demarcated from the background such as the corpus callosum, anterior and posterior commissures, optic chiasm, and cerebellar peduncles. Neuropathological studies can also detect absence of axons that normally cross the midline at many points in the brain stem and spinal cord, which are more difficult to visualize by standard magnetic resonance imaging (MRI). Electrophysiological studies such as evoked potentials can reveal aberrant central connections of peripheral sensory or motor nerves.The genetic disorders with aberrant axon connectivity presented in this article have been defined primarily using traditional approaches described above. Exciting advances in neuroimaging and genetics, however, are revolutionizing the ability to define axon guidance disorders, and it is likely that these syndromes are only the first of an important new category of such human neurodevelopmental disorders. Detailed fiber tract anatomy can now be visualized using noninvasive tractography such as diffusion tensor imaging (DTI) and diffusion spectrum imaging (DSI). These techniques provide tract orientation by determining the anisotropic properties of water diffusion, and can be used to reconstruct the trajectories of fiber systems in three-dimensional space (Tovar-Moll et al. 2007; Wahl et al. 2009). Tractography has successfully confirmed aberrant projections in several of the disorders discussed below (Fig. 2). At the same time, human genetics now provides an unbiased approach to identify the etiologies of disorders with aberrant axon tracts. For some syndromes, animal and in vitro studies have confirmed that the encoded protein has a primary role in axon guidance. For others, such studies reveal a primary role in neuronal specification and/or migration rather than, or in addition to, a role in axon guidance. Finally, some neurodevelopmental disorders without clinical, pathologic, or radiologic evidence of aberrant axon tracts have been found to result from mutations in genes that contribute to axon guidance in animal models.Open in a separate windowFigure 2.Tractography studies in patients with partial agenesis of the corpus callosum (pACC). T1-weighted anatomic images and DTI tractography of six subjects with pACC (top panels) and two representative controls (bottom panel). Axial (left) and midline sagittal (middle) T1 sections are shown for each subject. Callosal fragments are identified with yellow arrows, and heterotopic fibers visible on T1-weighted images are denoted by red arrows. Midline sagittal DTI color maps are shown with segmented callosal fibers (right). For subjects with pACC, connectivity ranged from anterior frontal connections (subject 3) to only posterior frontal and occipitotemporal connections (subject 4). One individual (subject 5) displayed a discontinuous set of homotopic callosal connections, with anterior frontal and occipitotemporal connectivity without any posterior frontal or parietal connections. Control subjects (bottom panel) display normal callosal morphology and tractography results. Tracts are segmented and colored according to their cortical projections: homotopic anterior frontal, blue; homotopic posterior frontal, orange; homotopic parietal, pink; homotopic occipitotemporal, green; heterotopic left anterior-right posterior, yellow; heterotopic right anterior-left posterior, red. (Reprinted, with permission, from Wahl et al. 2009 [© AJNR].)The major human genetic disorders that result, or are proposed to result, from defective axon guidance are ordered below from rostral to caudal based on the location of the aberrant axons tracts. These include genetic mutations that alter axon growth cone ligands and receptors, downstream signaling molecules, and axon transport, as well as proteins without currently recognized roles in axon guidance (Fig. 3) (Open in a separate windowFigure 3.Schematic representation of gene products implicated in human disorders of axon guidance. KAL1 (anosmin) and PROK2 are shown as secreted ligands. ROBO3, L1, and PROKR2 are shown as transmembrane receptors on the growth cone. CHN1 is depicted with 3 green domains (SH2, C1, RacGAP), responding to an unknown activated receptor and altering a microtubule, which is depicted as a brown line. KIF21A dimers are depicted walking down MTs. The OCA/OA and JSRD gene products are not depicted. Note: these gene products are not necessarily expressed in the same neurons or function in the same pathways.

Table 1

Summary of major human genetic disorders resulting, or hypothesized to result, from errors in axon growth and guidance

Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria

A number of methods have recently been published that use phylogenetic information extracted from large multiple sequence alignments to detect sites that have changed properties in related protein families. In this study we use such methods to assess functional divergence between eukaryotic EF-1α (eEF-1α), archaebacterial EF-1α (aEF-1α) and two eukaryote-specific EF-1α paralogs—eukaryotic release factor 3 (eRF3) and Hsp70 subfamily B suppressor 1 (HBS1). Overall, the evolutionary modes of aEF-1α, HBS1 and eRF3 appear to significantly differ from that of eEF-1α. However, functionally divergent (FD) sites detected between aEF-1α and eEF-1α only weakly overlap with sites implicated as putative EF-1β or aminoacyl-tRNA (aa-tRNA) binding residues in EF-1α, as expected based on the shared ancestral primary translational functions of these two orthologs. In contrast, FD sites detected between eEF-1α and its paralogs significantly overlap with the putative EF-1β and/or aa-tRNA binding sites in EF-1α. In eRF3 and HBS1, these sites appear to be released from functional constraints, indicating that they bind neither eEF-1β nor aa-tRNA. These results are consistent with experimental observations that eRF3 does not bind to aa-tRNA, but do not support the ‘EF-1α-like’ function recently proposed for HBS1. We re-assess the available genetic data for HBS1 in light of our analyses, and propose that this protein may function in stop codon-independent peptide release.     

A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

Background

Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy.

Methods

Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table ​(Table1)1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop.

Table 1

Patient Demographic Data

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Faunistic and bibliographical inventory of the Psychodinae moth-flies of North Africa (Diptera,Psychodidae)

All published records for the 49 species of moth flies known from North Africa are reviewed and discussed: Morocco (27 species), Algeria (33 species), Tunisia (18 species) and Egypt (five species). In addition, records of seven species of Psychodinae new to the fauna of Morocco are added, of which three are new mentions for North Africa (Table ​(Table1)1) and one is a new record for Egypt. Telmatoscopus squamifer Tonnoir, 1922 is transferred to the genus Iranotelmatoscopus Ježek, 1987, comb. n. Satchelliella reghayana Boumezzough & Vaillant, 1987 is transferred to the genus Pneumia Enderlein, 1935, comb. n. Pneumia aberrans Tonnoir, 1922 is transferred to the subgenus Logima.

Table 1.

Species (in alphabetical order) of Psychodinae known from the North African countries. Libya has been omitted because no information exists in the literature from Libya.

Association of SULT1A1 and UGT1A1 polymorphisms with breast cancer risk and phenotypes in Russian women

Estrogens are critical for breast cancer initiation and development. Sulfotransferase 1A1 (SULT1A1) and UDP-glucuronosyltransferase 1A1 (UGT1A1) conjugate and inactivate both estrogens and their metabolites, thus preventing estrogen-mediated mitosis and mutagenesis. SULT1A1 and UGT1A1 are both polymorphic, and different alleles encode functionally different allozymes. We hypothesize that low-activity alleles SULT1A1*2 and UGT1A1*28 are associated with higher risk for breast cancer and more severe breast tumor phenotypes. We performed a case-control study, which included 119 women of Russian ancestry with breast cancer and 121 age-matched Russian female controls. We used PCR followed by pyrosequencing to determine the SULT1A1 and UGT1A1 genotypes. Allele UGT1A1*28 was present at a higher frequency than the wild-type UGT1A1*1 allele in breast cancer patients as compared to controls (P = 0.002, OR = 1.79, CI 1.23–2.63). Consistently, the frequency of genotypes that contain allele UGT1A1*28 in the homozygous or the heterozygous state was greater in breast cancer patients as compared with the frequency of the wild-type UGT1A1*1/*1 genotype (P = 0.003, OR = 4.00, CI 1.49–11.11 and P = 0.014, OR = 2.04, CI 1.14–3.57, respectively). Individuals carrying allele UGT1A1*28 in the homo-or heterozygous state had larger breast tumors (>2 cm) as compared to the group with high-activity genotypes (P = 0.011, IR = 3.44, CI 1.42–8.36). No association was observed between any of the SULT1A1 genotypes and breast cancer risk or phenotypes. Our data suggest that UGT1A1, but not SULT1A1, genotypes are important for breast cancer risk and phenotype in Russian women. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 2, pp. 263–270. The article was translated by the authors.     

Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation

The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P < 0.05) in the percentage of live, caspase-activated spermatozoa (2.9 ± 0.7% vs 14.2 ± 6.4%; mean ± S.E.M.), low mitochondrial membrane potential (MMP; 6.8 ± 1.1 vs 23.8 ± 3.7), altered plasma membrane permeability (1.3 ± 0.2 vs 3.0 ± 0.5), DNA fragmentation (2.0 ± 1.3 vs 14.3 ± 3.6), total motility (81.8 ± 3.3 vs 35.1 ± 5.4), and progressive motility (66.3 ± 4.3 vs 24.1 ± 4.5) for high-density versus low-density subpopulations, respectively. Phosphatidylserine externalization did not differ (P = 0.67) between the high- and low-density subpopulations (2.6 ± 0.7 vs 3.1 ± 0.9). After cryopreservation, equine spermatozoa differed (P < 0.01) in the percentage of active caspases (19.1 ± 1.6 vs 52.1 ± 2.8), low MMP (18.2 ± 2.5 vs 48.7 ± 2.6), altered plasma membrane permeability (6.8 ± 1.7 vs 17.6 ± 2.0), total motility (75.5 ± 2.4 vs 45.2 ± 5.6), and progressive motility (53.9 ± 3.1 vs 28.3 ± 4.5) for pre-freeze versus cryopreserved spermatozoa. There was no difference (P = 0.21) in percentage of DNA fragmented cells before (5.5 ± 1.2) versus after cryopreservation (6.6 ± 1.1). We concluded that apoptotic-like changes were detectable in ejaculated equine spermatozoa and were more prevalent after cryopreservation.     

Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSP^WT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.     

Comparative Pathobiology of Kaposi Sarcoma-associated Herpesvirus and Related Primate Rhadinoviruses

With the emergence of the AIDS epidemic over the last 2 decades and the more recent identification of Kaposi sarcoma-associated herpesvirus (KSHV, Human herpesvirus 8), the genera of rhadinoviruses have gained importance as a family of viruses with oncogenic potential. First recognized in New World primates more than 30 y ago, the rhadinoviruses Saimiriine herpesvirus 2 and Ateline herpesvirus 2 have well-described transforming capabilities. Recently several new species-specific rhadinoviruses of Old World primates have been described, including retroperitoneal fibromatosis herpesvirus and rhesus rhadinovirus (Cercopithecine herpesvirus 17). Molecular analysis of these viruses has elucidated several functionally conserved genes and properties shared with KSHV involved in cellular proliferation, transformation, and immune evasion that facilitate the oncogenic potential of these viruses. This review examines the comparative pathobiology of KSHV, discusses the role of macaque rhadinoviruses as models of human disease, and outlines the derivation of specific pathogen-free animals.Abbreviations: CCL, cellular chemokine ligand; IRF, interferon regulatory factors; KSHV, Kaposi sarcoma-associated herpesvirus; LANA, latent nuclear antigen; MCD, multicentric Castleman disease; MCP1, monocyte chemotactic protein 1; miRNA, microRNA; ORF, open reading frame; PEL, primary effusion lymphoma; RFHV, retroperitoneal fibromatosis herpesvirus; RVV, rhesus rhadinovirus; SaHV2, Saimiriine herpesvirus 2; SPF, specific pathogen-free; SRV2, simian retrovirus type 2; THBS1, thrombospondinMembers of the herpesviridae are enveloped DNA viral agents that can infect a variety of host species, resulting in lifelong infection. The family is divided into Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, according to biologic behavior and phylogenetic relationship. As a group, synthesis of viral DNA occurs in the nucleus, and production of infectious virions is associated with destruction of the cell. Herpesviruses have large complex genomes and often have acquired host genes that allow these viruses to modulate and persist in the face of host immune responses.^25,71 This condition (termed ‘latency’) is characteristic of all herpesviral infections of the natural host. Although most members of the herpesviridae are of relatively low virulence in their respective hosts, some lack strict host specificity, and cross-species transmission to an inadvertent host can be associated with severe and fatal disease.The gammaherpesvirinae subfamily is characterized by in vitro and in vivo infection of lymphoblastoid cells and is further divided into the lymphocryptovirus (γ1 herpesviruses) and rhadinovirus (γ2 herpesviruses) genera. Rhadinoviruses have taken on increased importance with the identification of the novel Kaposi sarcoma-associated herpesvirus (KSHV, Human herpesvirus 8) in association with Kaposi sarcoma, an inflammatory and neoplastic condition seen in many HIV-infected patients with AIDS.^20,22 Until the recognition of KSHV more than a decade ago, rhadinovirus infection of primates was thought to be restricted to the New World primate lineages, but subsequent investigation revealed a number of novel species-specific viruses in a variety of Old World primates (28 As discussed later, based largely on phylogenetic analysis, it is now believed that the rhadinoviruses are subdivided into 2 distinct groupings (rhadinovirus [RV] 1 and 2).⁷⁷ This review will examine 2 recently recognized rhadinoviruses of macaques (retroperitoneal fibromatosis virus [RFHV] and rhesus rhadinovirus [RRV, Cercopethecine herpesvirus 17]), focusing on their comparative pathobiology with KSHV, their impact on naturally occurring disease entities, and their roles as animal models of human disease.

Table 1.

Nomenclature of primate rhadinoviruses (RV)

Mechanisms and Evidence of Genital Coevolution: The Roles of Natural Selection,Mate Choice,and Sexual Conflict

Genital coevolution between the sexes is expected to be common because of the direct interaction between male and female genitalia during copulation. Here we review the diverse mechanisms of genital coevolution that include natural selection, female mate choice, male–male competition, and how their interactions generate sexual conflict that can lead to sexually antagonistic coevolution. Natural selection on genital morphology will result in size coevolution to allow for copulation to be mechanically possible, even as other features of genitalia may reflect the action of other mechanisms of selection. Genital coevolution is explicitly predicted by at least three mechanisms of genital evolution: lock and key to prevent hybridization, female choice, and sexual conflict. Although some good examples exist in support of each of these mechanisms, more data on quantitative female genital variation and studies of functional morphology during copulation are needed to understand more general patterns. A combination of different approaches is required to continue to advance our understanding of genital coevolution. Knowledge of the ecology and behavior of the studied species combined with functional morphology, quantitative morphological tools, experimental manipulation, and experimental evolution have been provided in the best-studied species, all of which are invertebrates. Therefore, attention to vertebrates in any of these areas is badly needed.Of all the evolutionary interactions between the sexes, the mechanical interaction of genitalia during copulation in species with internal fertilization is perhaps the most direct. For this reason alone, coevolution between genital morphologies of males and females is expected. Morphological and genetic components of male and female genitalia have been shown to covary in many taxa (Sota and Kubota 1998; Ilango and Lane 2000; Arnqvist and Rowe 2002; Brennan et al. 2007; Rönn et al. 2007; Kuntner et al. 2009; Tatarnic and Cassis 2010; Cayetano et al. 2011; Evans et al. 2011, 2013; Simmons and García-González 2011; Yassin and Orgogozo 2013; and see examples in TaxaMale structuresFemale structuresEvidenceLikely mechanismReferencesMollusks Land snails (Xerocrassa)Spermatophore-producing organsSpermatophore-receiving organsComparative among speciesSAC or female choiceSauder and Hausdorf 2009 SatsumaPenis lengthVagina lengthCharacter displacementLock and keyKameda et al. 2009Arthropods Arachnids (Nephilid spiders)MultipleMultipleComparative among speciesSACKuntner et al. 2009 Pholcidae spidersCheliceral apophysisEpigynal pocketsComparative (no phylogenetic analysis)Female choiceHuber 1999 Harvestmen (Opiliones)Hardened penes and loss of nuptial giftsSclerotized pregenital barriersComparative among speciesSACBurns et al. 2013Millipedes Parafontaria tonomineaGonopod sizeGenital segment sizeComparative in species complexMechanical incompatibility resulting from Intersexual selectionSota and Tanabe 2010 Antichiropus variabilisGonopod shape and sizeAccesory lobe of the vulva and distal projectionFunctional copulatory morphologyLock and keyWojcieszek and Simmons 2012Crustacean Fiddler crabs, UcaGonopodeVulva, vagina, and spermathecaTwo-species comparison, shape correspondenceNatural selection against fluid loss, lock and key, and sexual selectionLautenschlager et al. 2010Hexapodes OdonatesClasping appendagesAbdominal shape and sensory hairsFunctional morphology, comparative among speciesLock and key via female sensory systemRobertson and Paterson 1982; McPeek et al. 2009Insects Coleoptera: seed beetlesSpiny aedagusThickened walls of copulatory ductComparative among speciesSACRönn et al. 2007 Callosobruchus: Callosobruchus maculatusDamage inflictedSusceptibility to damageFull sib/half sib mating experimentsSACGay et al. 2011Reduced spinesNo correlated responseExperimental evolutionSACCayetano et al. 2011 Carabid beetles (Ohomopterus)Apophysis of the endophallusVaginal appendix (pocket attached to the vaginal apophysis)Cross-species matingsLock and keySota and Kubota 1998; Sasabi et al. 2010 Dung beetle: Onthophagus taurusShape of the parameres in the aedagusSize and location of genital pitsExperimental evolutionFemale choiceSimmons and García-González 2011 Diptera: Drosophila santomea and D. yakubaSclerotized spikes on the aedagusCavities with sclerotized plateletsCross-species matingsSACKamimura 2012 Drosophila melanogaster species complexEpandrial posterior lobes
Oviscapt pouchesComparative among speciesSAC or female choiceYassin and Orgogozo 2013Phallic spikesOviscapt furrowsCercal teeth, phallic hook, and spinesUterine, vulval, and vaginal shields D. mauritiana and D. secheliaPosterior lobe of the genital archWounding of the female abdomenMating with introgressed linesSACMasly and Kamimura 2014 Stalk-eyed flies (Diopsidae)Genital processCommon spermathecal ductComparative among species and morphologicalFemale choiceKotrba et al. 2014 Tse-tse flies: Glossina pallidipesCercal teethFemale-sensing structuresExperimental copulatory functionFemale choiceBriceño and Eberhard 2009a,b Phelebotomine: sand fliesAedagal filaments, aedagal sheathsSpermathecal ducts length, base of the ductComparative among speciesNone specifiedIlango and Lane 2000 Heteroptera: Bed bugs (Cimiciidae)Piercing genitaliaSpermalege (thickened exosqueleton)Comparative among speciesSACCarayon 1966; Morrow and Arnqvist 2003 Plant bugs (Coridromius)Changes in male genital shapeExternal female paragenitaliaComparative among speciesSACTatarnic and Cassis 2010 Waterstriders (Gerris sp.)Grasping appendagesAntigrasping appendagesComparative among speciesSACArnqvist and Rowe 2002 Gerris incognitusGrasping appendagesAntigrasping appendagesComparative among populationsSACPerry and Rowe 2012 Bee assassins (Apiomerus)AedagusBursa copulatrixComparative among speciesNoneForero et al. 2013 Cave insects (Psocodea), NeotroglaMale genital chamberPenis-like gynosomeComparative among speciesFemale competition (role reversal), coevolution SACYoshizawa et al. 2014 Butterflies (Heliconiinae)Thickness of spermatophore wallSigna: Sclerotized structure to break spermatophoresComparative among speciesSACSánchez and Cordero 2014Fish Basking shark: Cetorhinus maximusClasper clawThick vaginal padsMorphological observationNoneMatthews 1950 GambusiaGonopodial tipsGenital papillae within openingsComparative among speciesStrong character displacementLangerhans 2011 Poecilia reticulataGonopodium tip shapeFemale gonopore shapeComparative among populationsSACEvans et al. 2011Reptiles AnolesHemipene shapeVagina shapeShape correspondence, two speciesSexual selectionKöhler et al. 2012 Several speciesHemipene shapeVagina shapeShape correspondenceLock and key, female choice, and SACPope 1941; Böhme and Ziegler 2009; King et al. 2009 Asiatic pit vipersSpininess in hemipenesThickness of vagina wallTwo-species comparisonNonePope 1941 Garter snake: Thamnophis sirtalisBasal hempene spineVaginal muscular controlExperimental manipulationSACFriesen et al. 2014Birds WaterfowlPenis lengthVaginal elaborationComparative among speciesSACBrennan et al. 2007 TinamousPenis length/presenceVaginal elaborationComparative among speciesFemale choice/natural selectionPLR Brennan, K Zyscowski, and RO Prum, unpubl.Mammals MarsupialsBifid penisTwo lateral vaginaeShape correspondenceNoneRenfree 1987 EquidnaBifid penis with four rosettesSingle vagina splits into two uteriShape correspondenceNoneAugee et al. 2006; Johnston et al. 2007 Insectivores: Short-tailed shrew: Blarina brevicaudaS-shaped curve of the erect penisCoincident curve in the vaginaShape correspondenceNoneBedford et al. 2004 Common tenrec: Tenrec caudatusFiliform penis (up to 70% of the male’s body length)Internal circular folds in the vaginaLength correspondenceNoneBedford et al. 2004 Rodents: Cape dune mole: Bathyergus suillusPenis and baculum lengthVaginal lengthAllometric relationships within speciesNoneKinahan et al. 2007 Australian hopping mice (Notomys)Spiny penisDerived distal region in the vaginaMorphological observation and two-species comparisonCopulatory lockBreed et al. 2013 Pig: Sus domesticusFiliform penis endCervical ridgesArtificial inseminationFemale choiceBonet et al. 2013 Primates: Macaca arctoidesLong and filamentous glansVestibular colliculus (fleshy fold) that partially obstructs the entrance to the vaginaShape correspondence and comparison with close relativesNoneFooden 1967Open in a separate windowThe likely mechanism is that suggested by the authors, and it includes sexually antagonistic coevolution (SAC), natural selection, sexual selection, female choice, or none specified. The evidence provided by the studies can be comparative among species or among populations, experimental evolution, cross-species matings, full-sibling (sib)/half-sib matings, shape, and length correspondence. Shape correspondence is often taken as evidence of coevolution, although it is not as conclusive as other approaches.Male genitalia are among the most variable structures in nature (Eberhard 1985). In contrast, female genitalia have typically been found not to be as interspecifically variable as male genitalia in several studies that specifically examined and described them (Eberhard 1985, 2010a,b). Female genitalia are not studied as often as male genitalia, perhaps because of a male-biased view of evolutionary processes by researchers (Ah-King et al. 2014). However, studying female genitalia is undeniably challenging. Male genitalia are generally kept inside of the body cavity, but are everted before, or during copulation, so their functional morphology can be more easily studied than the internal genitalia of females. Female genitalia also tend to be softer than male genitalia and thus their morphology may be more difficult to describe, and can more easily be distorted on dissection and preservation. Female adaptations to sense or oppose features of male genitalia can be subtle, requiring careful study. Female genital tracts are under multiple sources of selection: not just mating, but also storing sperm, egg laying, birthing, and often interfacing with the terminal portion of the digestive tract. Therefore, selection balancing multiple functions may further constrain morphological evolution in female genitalia. However, even small morphological changes in female genitalia, for example, increases in vaginal muscle, may change a female’s ability to choose or reject a male during mating, or to manage the costs of mating. Thus, the functional consequences to male and female genital morphology are hard to predict unless one knows how genitalia function during intromission. Despite these challenges, recent studies have examined variation of female genitalia and evidence is accumulating that features of female genitalia are variable enough to support coevolutionary processes (Polihronakis 2006; Puniamoorthy et al. 2010; Siegel et al. 2011; Showalter et al. 2013; and see additional references in Ah-King et al. 2014).In this article, we will discuss different hypotheses of genital evolution that predict coevolution; however, this is not a review of that entire subject (but see Eberhard et al. 2010b; Simmons 2013). Rather, we discuss the various mechanisms of genital coevolution differentiating the potentially independent or overlapping roles of natural selection, female choice, and male–male competition (Fig. 1). This classification allows us to distinguish specifically those mechanisms of genital coevolution that involve sexual conflict (i.e., when the evolutionary interests of individuals of different sexes, particularly over mating, are different). We then highlight examples in different taxa organisms with particular emphasis on those that provide evidence of sexual conflict.Open in a separate windowFigure 1.Graphical classification of mechanisms of genital evolution and coevolution. Three circles depict the independent and co-occurring actions of natural selection, female choice, and male–male competition. Different specific versions of genital coevolution can occur depending on which of the three broader evolutionary mechanisms are occurring. Sexual conflict (hatched lines) occurs through the simultaneous action of male–male competition and female choice, or male–male competition and natural selection. SAC, sexually antagonistic coevolution. See text for explanation.