Eco‐genetic additivity of diploids in allopolyploid wild wheats

Underpinnings of the distribution of allopolyploid species (hybrids with duplicated genome) along spatial and ecological gradients are elusive. As allopolyploid speciation combines the range of genetic and ecological characteristics of divergent diploids, allopolyploids initially show their additivity and are predicted to evolve differentiated ecological niches to establish in face of their competition. Here, we use four diploid wild wheats that differentially combined into four independent allopolyploid species to test for such additivity and assess the impact of ecological constraints on species ranges. Divergent genetic variation from diploids being fixed in heterozygote allopolyploids supports their genetic additivity. Spatial integration of comparative phylogeography and modelling of climatic niches supports ecological additivity of locally adapted diploid progenitors into allopolyploid species which subsequently colonised wide ranges. Allopolyploids fill suitable range to a larger extent than diploids and conservative evolution following the combination of divergent species appears to support their expansion under environmental changes.     

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.     

Missense substitutions reflecting regulatory control of transmitter phosphatase activity in two‐component signalling

Negative control in two‐component signal transduction results from sensor transmitter phosphatase activity for phospho‐receiver dephosphorylation. A hypothetical mechanism for this reaction involves a catalytic residue in the H‐box active‐site region. However, a complete understanding of transmitter phosphatase regulation is hampered by the abundance of kinase‐competent, phosphatase‐defective missense substitutions (K⁺ P^– phenotype) outside of the active‐site region. For the Escherichia coli NarX sensor, a model for the HisKA_3 sequence family, DHp domain K⁺ P^– mutants defined two classes. Interaction mutants mapped to the active site‐distal base of the DHp helix 1, whereas conformation mutants were affected in the X‐box region of helix 2. Thus, different types of perturbations can influence transmitter phosphatase activity indirectly. By comparison, K⁺ P^– substitutions in the HisKA sensors EnvZ and NtrB additionally map to a third region, at the active site‐proximal top of the DHp helix 1, independently identified as important for DHp‐CA domain interaction in this sensor class. Moreover, the NarX transmitter phosphatase activity was independent of nucleotides, in contrast to the activity for many HisKA family sensors. Therefore, distinctions involving both the DHp and the CA domains suggest functional diversity in the regulation of HisKA and HisKA_3 transmitter phosphatase activities.     

Hydrocarbon Repellents Isolated from Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae)

Seven unsaturated hydrocarbons were isolated from the volatiles of two species of flour beetles, Tribolium castaneum and T. confusum, and identified as 1-pentadecene, 1-heptadecene, 1,8-heptadecadiene, 1-tetradecene, 1-hexadecene, 1,6-pentadecadiene and heptadecatriene by GLC, GLC–MS, NMR, IR and micro-ozonolysis. This is the first report of the presence of these compounds in red flour beetles, and the last four compounds are also reported for the first time in confused flour beetles. These compounds are strong repellents for the same species or each other. The roles of these compounds are discussed.     

Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells

Fish, exposed to elevated water CO₂, experience a rapid elevation in blood CO₂ (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. White sturgeon, Acipenser transmontanus, are exceptionally CO₂ tolerant and can regulate tissue intracellular pH (pH_i) in the presence of a pronounced hypercapnic acidosis (preferential pH_i regulation). In this study, pH_i regulatory capacity of sturgeon liver cells in primary culture was examined to assess the suitability of employing this in vitro system to understand in vivo CO₂ tolerance in sturgeon. Using the pH-sensitive fluoroprobe BCECF, real-time changes in resting pH_i and rates of pH_i recovery were investigated during exposure to hypercapnia (3 and 6% CO₂) in the absence and presence of additional acid loads induced by (20 mM) ammonium prepulse. During short-term (10 min) exposure to hypercapnia (3 and 6% CO₂), sturgeon cells were acidified and no pH_i compensation was observed. However, when exposure to 6% CO₂ was extended to over 19 h, the CO₂-induced intracellular acidosis was partially compensated by a pH_i increase of over 0.2 pH unit despite the sustained extracellular acidosis, indicative of a capacity for preferential pH_i regulation in vitro. Since this capacity in sturgeon liver is present both in vivo and in vitro, the transmembrane transporters involved may be the same. Therefore, cell culture may be a suitable tool to identify the transporters (i.e., the cellular mechanisms underlying in vivo CO₂ tolerance) in white sturgeon and possibly in other hypercapnia-tolerant species.     

Identification of residues within tropomodulin-1 responsible for its localization at the pointed ends of the actin filaments in cardiac myocytes

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.     

Brain circuits for mating behavior in cats and brain activations and de-activations during sexual stimulation and ejaculation and orgasm in humans

In cats, there exists a descending system that controls the posture necessary for mating behavior. A key role is played by the mesencephalic periaqueductal gray (PAG), which maintains strong specific projections to the nucleus retroambiguus located laterally in the most caudal medulla. The NRA, in turn, has direct access to motoneurons in the lumbosacral cord that produce the mating posture. This pathway is slightly different in males and females, but in females its strength fluctuates strongly depending on whether or not the cat is in heat. This way the PAG determines whether or not mating can take place. Via the PAG many other regions in the limbic system as well as in the prefrontal cortex and insula can influence mating behavior.In humans, the brain also controls responses to sexual stimulation as well as ejaculation in men and orgasm in women. Neuroimaging techniques show activations and de-activations but are not able to verify whether the PAG has a similar effect as in cats. PET-scanning results revealed that there is activation in the upper brainstem and cerebellum, as well as insula in men and in the somatomotor and somatosensory cortex in women. During sexual stimulation, but especially during ejaculation and orgasm there was strong de-activation mainly on the left side in the temporal lobe and ventral prefrontal cortex. These neuroimaging results show the importance of lowering the level of alertness regarding your immediate environment (left hemisphere) to have proper sexual behavior.     

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.     

Reversible covalent inhibition of a phenol sulfotransferase by coenzyme A

Phenol sulfotransferases (SULTs), which normally bind 3'-phosphoadenosine-5'-phosphosulfate as the donor substrate, are inhibited by CoA and its thioesters. Here, we report that inhibition of bovine SULT1A1 by CoA is time-dependent at neutral pH under non-reducing conditions. The rates of inactivation by CoA indicate an initial reversible SULT:CoA complex with a dissociation constant of 5.7 microM and an inactivation rate constant of 0.07 min(-1). Titrations with CoA and prolonged incubations reveal that inactivation of the dimeric enzyme is stoichiometric, consistent with the observation of complete conversion of the protein to a slightly decreased electrophoretic mobility. Both activity and normal electrophoretic migration are restored by 2-mercaptoethanol. Mutagenesis demonstrated that Cys168 is the site of CoA adduction, and a consistent model was constructed that reveals a new SULT molecular dynamic. Cysteine reaction kinetics with Ellman's reagent revealed a PAPS-induced structural change consistent with the model that accounts for binding of CoA.