Contrasting geographic patterns of genetic variation for molecular markers vs. phenotypic traits in the energy grass Miscanthus sinensis

Species and hybrids of Miscanthus are a promising energy crop, but their outcrossing mating systems and perennial life cycles are serious challenges for breeding programs. One approach to accelerating the domestication of Miscanthus is to harness the tremendous genetic variation that is present within this genus using phenotypic data from extensive field trials, high‐density genotyping and sequencing technologies, and rapidly developing statistical methods of relating phenotype to genotype. The success of this approach, however, hinges on detailed knowledge about the population genetic structure of the germplasm used in the breeding program. We therefore used data for 120 single‐nucleotide polymorphism and 52 simple sequence repeat markers to depict patterns of putatively neutral population structure among 244 Miscanthus genotypes grown in a field trial near Aberystwyth (UK) and delineate a population of 145 M . sinensis genotypes that will be used for association mapping and genomic selection. Comparative multivariate analyses of molecular marker and phenotypic data for 17 traits related to phenology, morphology/biomass, and cell wall composition revealed significant geographic patterns in this population. A longitudinal cline accounted for a substantial proportion of molecular marker variation (R² = 0.60, P = 3.4 × 10^?15). In contrast, genetic variation for phenotypic traits tended to follow latitudinal and altitudinal gradients, with several traits appearing to have been affected by divergent selection (i.e., Q_ST >> F_ST). These contrasting geographic trends are unusual relative to other plants and provide opportunities for powerful studies of phenotype–genotype associations and the evolutionary history of M. sinensis.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

The Structure of an Interdomain Complex That Regulates Talin Activity

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1) and in coupling integrins to the actin cytoskeleton (2). Thus, depletion of talin results in defects in integrin activation (3), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4). In the whole organism, studies on the single talin gene in worms (5) and flies (6) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8, 9), defects in the membrane-cytoskeletal interface in megakaryocytes (10), and disruption of the myotendinous junction in skeletal muscle (11). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12).Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The talin head contains a FERM² domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2). Studies by Wegener et al. (30) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin β-subunits (13). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin α- and β-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activation (14) may be explained by our recent finding that F0 binds, albeit with low affinity, Rap1-GTP,³ a known activator of integrins (15, 16). The talin rod is made up of a series of amphipathic α-helical bundles (17–20) and contains a second integrin binding site (IBS2) (21), numerous binding sites for the cytoskeletal protein vinculin (22), at least two actin binding sites (23), and a C-terminal helix that is required for assembly of talin dimers (20, 24).Both biochemical (25) and cellular studies (16) suggest that the integrin binding sites in full-length talin are masked, and both phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in exposing these sites. It is well established that some members of the FERM domain family of proteins are regulated by a head-tail interaction (26); gel filtration, sedimentation velocity, and electron microscopy studies all show that talin is globular in low salt buffers, although it is more elongated (∼60 nm in length) in high salt (27). By contrast, the talin rod liberated from full-length talin by calpain-II cleavage is elongated in both buffers, indicating that the head is required for talin to adopt a more compact state. Direct evidence for an interaction between the talin head and rod has recently emerged from NMR studies by Goksoy et al. (28), who demonstrated binding of ¹⁵N-labeled talin F3 to a talin rod fragment spanning residues 1654–2344, an interaction that was confirmed by surface plasmon resonance (K_d = 0.57 μm) (28). Chemical shift data also showed that this segment of the talin rod partially masked the binding site in F3 for the membraneproximal helix of the β3-integrin tail (28), directly implicating the talin head-rod interaction in regulating the integrin binding activity of talin. Goksoy et al. (28) subdivided the F3 binding site in this rod fragment into two sites with higher affinity (K_d ∼3.6 μm; residues 1654–1848) and lower affinity (K_d ∼78 μm; residues 1984–2344). Here, we define the rod domain boundaries and determine the NMR structure of residues 1655–1822, a five-helix bundle. We further show that this domain binds F3 predominantly via surface-exposed residues on helix 4, with an affinity similar to the high affinity site reported by Goksoy et al. (28). We also report the structure of the complex between F3 and the rod domain and show that the latter masks the known binding site in F3 for the β3-integrin tail and is expected to inhibit the association of the talin FERM domain with the membrane.     

Effects of systemic pancreastatin on memory retention

Pancreastatin, a peptide isolated from the pancreas, was shown to enhance memory retention after peripheral administration in mice when administration following T-maze footshock avoidance training. The effect of pancreastatin on memory retention, one week after training, was time dependent showing enhancement of retention when pancreastatin was administered 0 and 30 min but not 60 min after training. Pancreastatin reversed the amnesia produced by scopolamine. The pancreastatin fragment (33-49) also enhanced memory. Pancreastatin did not increase glucose in vivo. We conclude that peripherally administered pancreastatin modulates memory processing.     

Design and optimisation of orally active TLR7 agonists for the treatment of hepatitis C virus infection

The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.     

Reproductive investment and parental roles in Sabine's gulls<Emphasis Type="Italic"> Xema sabini</Emphasis>

More than 90% of avian species exhibit biparental care, though parental activities are often shared unequally between the members of a pair. Among gull species (Laridae), males and females generally share parental activities, although there appear to be considerable differences between species in the relative contribution of each sex. This study examined the behaviour of male and female Sabine's gulls (Xema sabini) during the incubation period and immediately post-hatch; particularly the amount of time they each invested in breeding activities. Although considered an aberrant species in aspects of behaviour and biology, the Sabine's gull showed a high reproductive investment by both sexes, as other gull species do. Males fed females prior to egg laying and contributed equally to incubation and chick provisioning, and females contributed equally to nest defence. Overall, there was no difference between the sexes in the extent of their contributions to parental care, although there was considerable individual variation within pairs. Sabine's gulls are Arctic breeders and the extent of their contributions to parental activities could have been influenced by their extreme breeding environment and short breeding season.     

From Rapid Place Learning to Behavioral Performance: A Key Role for the Intermediate Hippocampus

Rapid place encoding by hippocampal neurons, as reflected by place-related firing, has been intensely studied, whereas the substrates that translate hippocampal place codes into behavior have received little attention. A key point relevant to this translation is that hippocampal organization is characterized by functional–anatomical gradients along the septotemporal axis: Whereas the ability of hippocampal neurons to encode accurate place information declines from the septal to temporal end, hippocampal connectivity to prefrontal and subcortical sites that might relate such place information to behavioral-control processes shows an opposite gradient. We examined in rats the impact of selective lesions to relevant parts of the hippocampus on behavioral tests requiring place learning (watermaze procedures) and on in vivo electrophysiological models of hippocampal encoding (long-term potentiation [LTP], place cells). We found that the intermediate hippocampus is necessary and largely sufficient for behavioral performance based on rapid place learning. In contrast, a residual septal pole of the hippocampus, although displaying intact electrophysiological indices of rapid information encoding (LTP, precise place-related firing, and rapid remapping), failed to sustain watermaze performance based on rapid place learning. These data highlight the important distinction between hippocampal encoding and the behavioral performance based on such encoding, and suggest that the intermediate hippocampus, where substrates of rapid accurate place encoding converge with links to behavioral control, is critical to translate rapid (one-trial) place learning into navigational performance.     

Phenotypic and Genotypic Description of Sedimenticola selenatireducens Strain CUZ,a Marine (Per)Chlorate-Respiring Gammaproteobacterium,and Its Close Relative the Chlorate-Respiring Sedimenticola Strain NSS

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30°C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42°C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.     

Virtual resource development in the glycosciences

The development of Internet-based virtual resources is a relatively new area of scientific and technical activity that is currently undergoing rapid expansion. Major factors fuelling recent growth include the emergence of multimedia capabilities through the rapid evolution of the World Wide Web, the reduction in cost of high quality personal computers and graphics workstations and the provision of mass-marketed provider services. Prior to 1995 the presence of Internet resources in the glycosciences was virtually non-existent. Existing scientific knowledge was primarily made available on the Net through the provision of databases from gopher and ftp sites. A particular example in the glycosciences is the Carbbank database of biological carbohydrate sequences. We will describe here our efforts in 1994–95 in establishing The Glycoscience Network (TGN, http://bellatrix.pcl.ox.ac.uk/TGN/). These activities included the establishment of a newsgroup, mailing lists, Web resources and the running of the First Electronic Glycoscience Conference (EGC-1, http://bellatrix.pcl.ox.ac.uk/egc/). EGC-1 included many novel initiatives in the glycosciences including electronic posters and papers, a Virtual Conference Centre, a Web-based hyperglossary, Virtual Trade and Employment Centres, refereed electronic publishing, and the creation of a Virtual Reality Gallery. We would like to look towards the near future and discuss several initiatives in virtual resource creation that we believe will have significant scientific impact on the glycosciences including the development of bioinformatics-based servers, sophisticated interactive databases, and videoconferencing. Furthermore, we cherish the belief that these resources will foster international scientific collaboration and progress of an extent never previously possible. Finally, we indulge in speculation and make some suggestions on the form and long-term impact of Glycoscience Virtual Resources. We predict that their development may completely reconstruct the scientific environment that we work in as scientists and we reflect on the probable benefits and pitfalls to be encountered.This paper was presented at the First Electronic Glycoscience Conference (EGCI) on the World Wide Web, September 1995.     

Leptin and neuropeptide Y (NPY) modulate nitric oxide synthase: further evidence for a role of nitric oxide in feeding.

Recent studies have suggested a role for nitric oxide in the regulation of food intake. Neuropeptide Y (NPY) is one of the most potent orexigenic agents. Chronic administration of leptin decreases food intake. This study examined the effects of NPY and leptin on nitric oxide synthase (NOS) in the hypothalamus. Previously it has been demonstrated that obese (ob/ob) mice have elevated NOS levels in the hypothalamus. In this study we demonstrated that the administration of leptin (6 microg/day) subcutaneously (SC) for 3 days decreased body weight (P < 0.001) and food intake P < 0.001) in obese (ob/ob) mice as expected. In addition, leptin decreased NOS in the hypothalamus nu 37% (P < 0.01) and in brown adipose tissue by 69% (P < 0.01) but not in white adipose tissue. NPY was administered intracerebroventricularly to CD-1 mice at doses of 0.25 and 0.50 microg. Mice were sacrificed 15 min after injection and NOS was measured in their hypothalami. NPY at the lower dose increased NOS in the hypothalamus by 147%. These results, taken together, with previously published studies support the concept that nitric oxide may play a role as a mediator of the effects of NPY and leptin on food intake. The alterations of NOS in brown adipose tissue following leptin administration could result in changes in blood flow or metabolism in the brown fat.