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 mechanical significance of the occlusal geometry of great ape molars in food breakdown

Analyses of dental function are an essential component of the study of human evolution. However, with few exceptions, they have utilized the traditional analogizing method of comparative anatomy, and have assumed rather than demonstrated that proposed adaptive characters confer a performance benefit. Since food reduction is a mechanical process, it is appropriate to measure performance using mechanical parameters, specifically the ability of a given morphology to induce failure in food particle by either of the two major regimes: crush and shear, corresponding to simple stresses (tensile and compressive) and shear stress, respectively. We apply finite elements stress analysis to model the relationship between the angulation of the intercuspal occlusal surfaces in a “puncture crushing” mode of mastication. On the basis of morphological data acquired from sectioned great ape molars, we have predicted the nature, magnitude and distribution of stress in a standard food particle by models representing each morphotype. Results indicate that the blunt-cusped molars ofHomo, the gradually-sloping supporting (buccal) cusps but high-angled guiding (lingual) cusps of the lower molars ofPan, and the high angled occlusal surfaces ofGorillaare all more likely to fracture small food particles by shear, while the gradually sloping occlusal surfaces ofPongomolars are more likely to break them down by “crush”. Mechanisms of food failure induced by molars ofPanandHomowill vary according to the orientation of the tooth–food contacting surfaces, which in turn will vary according to the size of the food particle. These genera may be able to break food down either by shear or by “crush”.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Dual ultraviolet wavelength high-performance liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and some selected metabolites

A sensitive method suitable for the determination of tricyclic and other antidepressants in postmortem and clinical specimens is presented. The procedure, which utilizes reversed-phase HPLC combined with dual ultraviolet wavelength detection, enables the separation of 17 commonly prescribed antidepressants and some selected metabolites in a single extraction. Peak purity was confirmed using absorbance ratios at 220 nm and 254 nm wavelengths and revealed little interference from other eluting analytes. The blood detection limit for most antidepressants was 50 ng/ml. The most commonly observed antidepressants in 281 forensic cases analysed over a two-year period with the described method were dothiepin, amitriptyline, nortriptyline and doxepin.     

Identifying Mozambique's most critical areas for plant conservation: An evaluation of protected areas and Important Plant Areas

Successful protected area networks must represent biodiversity across taxonomic groups. However, too often plant species are overlooked in conservation planning, and the resulting protected areas may, as a result, fail to encompass the most important sites for plant diversity. The Mozambique Tropical Important Plant Areas project sought to promote the conservation of Mozambique's flora through the identification of Important Plant Areas (IPAs). Here, we use the Weighted Endemism including Global Endangerment (WEGE) index to identify the richest areas for rare and endemic plants in Mozambique and subsequently evaluate how well represented these hotspots are within the current protected area and IPA networks. We also examine the congruence between IPA and protected areas to identify opportunities for strengthening the conservation of plants in Mozambique. We found that high WEGE scores, representing areas rich in endemic/near-endemic and threatened species, predict the presence of IPAs in Mozambique, but do not predict the presence of protected areas. We also find that there is limited overlap between IPAs and protected areas in Mozambique. We demonstrate how IPAs could be an important tool for ensuring priority sites for plant diversity are included within protected area network expansions, particularly following the adoption of the “30 by 30” target agreed within the post-2020 Convention on Biological Diversity framework, with great potential for this method to be replicated elsewhere in the global tropics.     

Novel Miscanthus hybrids: Modelling productivity on marginal land in Europe using dynamics of canopy development determined by light interception

New biomass crop hybrids for bioeconomic expansion require yield projections to determine their potential for strategic land use planning in the face of global challenges. Our biomass growth simulation incorporates radiation interception and conversion efficiency. Models often use leaf area to predict interception which is demanding to determine accurately, so instead we use low-cost rapid light interception measurements using a simple laboratory-made line ceptometer and relate the dynamics of canopy closure to thermal time, and to measurements of biomass. We apply the model to project the European biomass potentials of new market-ready hybrids for 2020–2030. Field measurements are easier to collect, the calibration is seasonally dynamic and reduces influence of weather variation between field sites. The model obtained is conservative, being calibrated by crops of varying establishment and varying maturity on less productive (marginal) land. This results in conservative projections of miscanthus hybrids for 2020–2030 based on 10% land use conversion of the least (productive) grassland and arable for farm diversification, which show a European potential of 80.7–89.7 Mt year⁻¹ biomass, with potential for 1.2–1.3 EJ year⁻¹ energy and 36.3–40.3 Mt year⁻¹ carbon capture, with seeded Miscanthus sacchariflorus × sinensis displaying highest yield potential. Simulated biomass projections must be viewed in light of the field measurements on less productive land with high soil water deficits. We are attempting to model the results from an ambitious and novel project combining new hybrids across Europe with agronomy which has not been perfected on less productive sites. Nevertheless, at the time of energy sourcing issues, seed-propagated miscanthus hybrids for the upscaled provision of bioenergy offer an alternative source of renewable energy. If European countries provide incentives for growers to invest, seeded hybrids can improve product availability and biomass yields over the current commercial miscanthus variety.     

Evaluation of somatic embryos of interior spruce. Characterization and developmental regulation of storage proteins

Storage proteins of interior spruce ( Picea glauca engelmanii complex) somatic embryos were compared to those of zygotic embryos by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Somatic embryos contain the same storage proteins as zygotic embryos based on similarities of molecular weight, isoelectric variants, solubility characteristics and disulfide linkages. Storage protein levels varied among different somatic embryo genotypes; however, all genotypes tested accumulated significant amounts of storage proteins. Zygotic and somatic embryos display a similar developmental accumulation of storage proteins. The 22, 24, 33 and 35 kDa proteins appear in early stage embryos, while the 41 kDa protein begins to accumulate during mid cotyledon development. The 22, 24 and 41 kDa proteins accumulate continuously during cotyledon development in somatic embryos cultured on abscisic acid. In contrast, zygotic embryos display a more rapid and transient accumulation of these proteins.