Type II beta-turn conformation of pivaloyl-L-prolyl-alpha-aminoisobutyryl-N-methylamide: Theoretical, spectroscopic, and X-ray studies

Pivaloyl-L-Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD3)2SO solution, by 1H-nmr methods, suggesting the existence of beta -turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II beta-turn conformations are about 2 kcal mol-1 more stable than Type III structures. A crystallographic study has established the Type II beta-turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, beta = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II -turn conformation is stabilized by an intramolecular 4 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are Pro = -57.8°, Pro = 139.3°, Aib = 61.4°, and Aib = 25.1°. The Type II beta-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.

beta.-Turn conformation of N-acetyl-L-prolylglycyl-L-phenylalanine. Crystal structure and solution studies

Pro-Gly segments in peptides and proteins are prone to adopt the 0-turn conformation. This paper reports experimental data for the presence of this conformation in a linear tripeptide N-acetyl-L-prolylglycyl-L-phenylalanineb oth in the solid state and in solution. X-ray diffraction data on the tripeptide crystal show that it exists in the type I1 0-turn conformation. CD and proton NMR data show that this conformation persists in trifluoroethanol and methanol solutions in equilibrium with the nonhydrogen-bonded structures. Isomerization around the acetyl-prolyl bond is seen to take place in dimethyl sulfoxide solutions of the tripeptide.

Molecular vibrations, electronic structure and conformation of diprotonated thiocarbohydrazide

The infrared spectra of diprotonated species of thiocarbohydrazide and its perdeuterated derivative have been examined in the crystalline state. A complete vibrational assignment with a full normal coordinate treatment based on a Urey—Bradley type intramolecular potential Function supplemented with a valence force function for the out of plane and torsional modes is proposed and the origin of the amide II band splittings is explained. A CNDO/2 study of diprotonated thiocarbohydrazide and its neutral molecule is undertaken and the changes in the molecular electronic structures and conformations consequent to protonation are determined and briefly discussed. The magnitude of the N—N+H3 torsional barrier is estimated to be 21 kJ mol− (5.0 kcal mol−1) whereas the barrier for the C—N group is found to be 92 kJ mol−1 (22.0 kcal mol−1).

Computer-aided analysis of the structure of kinematic chains

Using the link-link incidence matrix to represent a simple-jointed kinematic chain algebraic procedures have been developed to determine its structural characteristics such as the type of freedom of the chain, the number of distinct mechanisms and driving mechanisms that can be derived from the chain. A computer program incorporating these graph theory based procedures has been applied successfully for the structural analysis of several typical chains.

Risk factors associated with corneal nerve alteration in type 1 diabetes in the absence of neuropathy: A longitudinal in vivo corneal confocal microscopy study

Purpose The aim of this study was to determine alterations to the corneal subbasal nerve plexus (SNP) over four years using in vivo corneal confocal microscopy (IVCM) in participants with type 1 diabetes and to identify significant risk factors associated with these alterations. Methods A cohort of 108 individuals with type 1 diabetes and no evidence of peripheral neuropathy at enrollment underwent laser-scanning IVCM, ocular screening, and health and metabolic assessment at baseline and the examinations continued for four subsequent annual visits. At each annual visit, eight central corneal images of the SNP were selected and analyzed to quantify corneal nerve fiber density (CNFD), branch density (CNBD) and fiber length (CNFL). Linear mixed model approaches were fitted to examine the relationship between risk factors and corneal nerve parameters. Results A total of 96 participants completed the final visit and 91 participants completed all visits. No significant relationships were found between corneal nerve parameters and time, sex, duration of diabetes, smoking, alcohol consumption, blood pressure or BMI. However, CNFD was negatively associated with HbA1c (β=-0.76, P<0.01) and age (β=-0.13, P<0.01) and positively related to high density lipids (HDL) (β=2.01, P=0.03). Higher HbA1c (β=-1.58, P=0.04) and age (β=-0.23, P<0.01) also negatively impacted CNBD. CNFL was only affected by higher age (β=-0.06, P<0.01). Conclusions Glycemic control, HDL and age have significant effects on SNP structure. These findings highlight the importance of diabetic management to prevent corneal nerve damage as well as the capability of IVCM for monitoring subclinical alterations in the corneal SNP in diabetes.

The Crystal Structure of Pivaloyl- D- prolyl-L- prolyl- L-alanyl-N- methylamide

Pivaloyl-D-prolyl-L-prolyl-L-analyl-N-methylam~de (I), C1UH32N40c4r,y stallizes in the orthorhombic space group P21212,w ith four molecules in a unit cell of dimensions a = 9.982 (l),b = 10.183 (3), c = 20.746 (2)A . The structure has been refined to R 0.048 for 1 745 observed reflections. All the peptide bonds in the molecule are trans and both the prolyl residues are in the CY-exo-conformation. The molecule assumes a highly folded conformation in which a Type II' DL bend is followed by a Type I LL bend, both stabilised by intramolecular 4 + 1 hydrogen bonds. This conformation, which has been observed for the first time, is of interest in relation to the structure of gramicidin S.

Population structure of Pyrenophora teres, the causal agent of net blotch of barley

Spring barley is the most important crop in Finland based on cultivated land area. Net blotch, a disease caused by Pyrenophora teres Drech., is the most damaging disease of barley in Finland. The pressure to improve the economics and efficiency of agriculture has increased the need for more efficient plant protection methods. Development of durable host-plant resistance to net blotch is a promising possibility. However, deployment of disease resistant crops could initiate selection pressure on the pathogen (P. teres) population. The aim of this study was to understand the population biology of P. teres and to estimate the evolutionary potential of P. teres under selective pressure following deployment of resistance genes and application of fungicides. The study included mainly Finnish P. teres isolates. Population samples from Russia and Australia were also included. Using AFLP markers substantial genotypic variation in P. teres populations was identified. Differences among isolates were least within Finnish fields and significantly higher in Krasnodar, Russia. Genetic differentiation was identified among populations from northern Europe and from Australia, and between the two forms P. teres f. teres (PTT, net form of net blotch) and P. teres f. maculata (PTM, spot form of net blotch) in Australia. Differentiation among populations was also identified based on virulence between Finnish and Russian populations, and based on prochloraz (fungicide) tolerance in the Häme region in Finland. Surprisingly only PTT was recovered from Finland and Russia although both forms were earlier equally common in Finland. The reason for the shift in occurrence of forms in Finland remained uncertain. Both forms were found within several fields in Australia. Sexual reproduction of P. teres was supported by recover of both mating types in equal ratio in those areas although the prevalence of sexual mating seems to be less in Finland than in Australia. Population from Krasnodar was an exception since only one mating type was found in there. Based on the substantial high genotypic variation in Krasnodar it was suggested go represent an old P. teres population, whereas the Australian samples were suggested to represent newer populations. In conclusion, P. teres populations are differentiated at several levels. Human assistance in dispersal of P. teres on infected barley seed is obvious and decreases the differentiation among populations. This can increase the plant protection problems caused by this pathogen. P. teres is capable of sexual reproduction in several areas but the prevalence varies. Based on these findings it is apparent that P. teres has the potential to pose more serious problems in barley cultivation if plant protection is neglected. Therefore, good agricultural practices, including crop rotation and the use of healthy seed, are recommended.

Microbial indicators related to yield and disease and changes in soil microbial community structure with ginger farm management practices

TRFLP (terminal restriction fragment length polymorphism) was used to assess whether management practices that improved disease suppression and/or yield in a 4-year ginger field trial were related to changes in soil microbial community structure. Bacterial and fungal community profiles were defined by presence and abundance of terminal restriction fragments (TRFs), where each TRF represents one or more species. Results indicated inclusion of an organic amendment and minimum tillage increased the relative diversity of dominant fungal populations in a system dependant way. Inclusion of an organic amendment increased bacterial species richness in the pasture treatment. Redundancy analysis showed shifts in microbial community structure associated with different management practices and treatments grouped according to TRF abundance in relation to yield and disease incidence. ANOVA also indicated the abundance of certain TRFs was significantly affected by farming system management practices, and a number of these TRFs were also correlated with yield or disease suppression. Further analyses are required to determine whether identified TRFs can be used as general or soil-type specific bio-indicators of productivity (increased and decreased) and Pythium myriotylum suppressiveness.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria , Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. © 2014 Penton et al.

Influence of Sm2O3 doping on formation and structure of SrBi2Nb2O9 nanocrystals in lithium borate glasses

New glasses of 16.66SrO–16.66[(1 − x)Bi2O3–xSm2O3]–16.66Nb2O5–50Li2B4O7 (0 ≤ x ≤ 0.5, in molar ratio), i.e., the pseudo-binary Sm2O3-doped SrBi2Nb2O9–Li2B4O7 glass system, giving the crystallization of Sm3+-doped SrBi2Nb2O9 nanocrystals are developed. It is found that the thermal stability of the glasses against the crystallization and the optical band gap energy increases with increasing Sm2O3 content. The formation of fluorite-type Sm3+-doped SrBi2Nb2O9 nanocrystals (diameters: 13–37 nm) with a cubic structure is confirmed in the crystallized (530 °C, 3 h) samples from X-ray powder diffraction analyses, Raman scattering spectrum measurements, and transmission electron microscope observations. The effect of Sm3+-doping on the microstructure, Raman scattering peak positions, and dielectric properties of composites comprising of fluorite-type SrBi2Nb2O9 nanocrystals and the Li2B4O7 glassy phase is clarified. It is found that fluorite-type SrBi2Nb2O9 nanocrystals transform to stable perovskite-type SrBi2Nb2O9 crystals with an orthorhombic structure by heat treatments at around 630 °C.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Boundedness and convergence of singular integrals on fractal type sets

The topic of this dissertation lies in the intersection of harmonic analysis and fractal geometry. We particulary consider singular integrals in Euclidean spaces with respect to general measures, and we study how the geometric structure of the measures affects certain analytic properties of the operators. The thesis consists of three research articles and an overview. In the first article we construct singular integral operators on lower dimensional Sierpinski gaskets associated with homogeneous Calderón-Zygmund kernels. While these operators are bounded their principal values fail to exist almost everywhere. Conformal iterated function systems generate a broad range of fractal sets. In the second article we prove that many of these limit sets are porous in a very strong sense, by showing that they contain holes spread in every direction. In the following we connect these results with singular integrals. We exploit the fractal structure of these limit sets, in order to establish that singular integrals associated with very general kernels converge weakly. Boundedness questions consist a central topic of investigation in the theory of singular integrals. In the third article we study singular integrals of different measures. We prove a very general boundedness result in the case where the two underlying measures are separated by a Lipshitz graph. As a consequence we show that a certain weak convergence holds for a large class of singular integrals.

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Higher-resolution structure of the human insulin receptor ectodomain: Multi-modal inclusion of the insert domain

Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.