Genetic engineering offers the potential for creating new proteins with novel materials properties. The aim of this study is to develop strategies for incorporating the non-natural amino acid ...p-fluorophenylalanine (pfF), which is known to be utilized by the Escherichia coli protein synthesis apparatus, into repetitive polypeptides. pfF has been successfully incorporated into target protein by in vivo substitution of pfF for phenylalanine in a phenylalanine auxotroph of the E. coli BL21(DE3) expression strain containing a chromosomal copy of the bacteriophage T7 RNA polymerase gene under LacUV5 promoter control. The auxotroph was generated by transposon mutagenesis using P1-mediated transduction. The repetitive protein -(Ala multiplied by Gly) sub(3) multiplied by pfF multiplied by Gly sub(13)- (1) was expressed from an artificial gene under T7 promoter control. Expression of 1 was carried out by shifting the host culture to pfF-containing medium after induction of T7 RNA polymerase for 10 min with isopropyl beta -D-thiogalactopyranoside in medium containing the 20 natural amino acids. Replacement of 95-100% of phenylalanine by pfF was confirmed by H NMR spectroscopy and amino acid analysis. Fourier transform infrared spectroscopy and X-ray scattering suggest that both 1 and the phenylalanine variant (2) adopt antiparallel beta -sheet structures in the solid state.
The crystal structures and textures of a family of sequence-designed periodic polypeptides were investigated and analyzed using X-ray diffraction, vibrational spectroscopy, and cross-polarization ...magic angle spinning 13C nuclear magnetic resonance. The repetitive amino acid sequences are described by −(AG) x EG−, with integer x from 3 to 6. These macromolecules were prepared via bacterial expression of artificial genes and are monodisperse. Crystalline samples were obtained, and the interpretation of the X-ray diffraction results was aided by the generation of computer-simulated X-ray diffraction patterns. This allowed direct comparisons to be made with the observed texture-oriented X-ray diffraction photographs. All diffraction and spectroscopic evidence supports an antiparallel (ap) β-sheet structure, and all structures index on orthorhombic sublattices similar to those reported for Bombyx mori silk fibroin and poly(l-alanylglycine). The unit cell parameters for poly(AG)3EG, for example, are a = 0.948 nm (hydrogen-bond direction), b = 1.060 nm (ap β-sheet stacking direction), and c = 0.695 nm (chain direction). Selective line broadening is observed for wide-angle diffraction signals with l ≠ 0 (for the 211 in particular) and gives an estimated crystal size of <4 nm in the chain direction. This, coupled with the appearance of a low-angle particle interference peak at 3.6 nm, indicates a crystal size over an order of magnitude less than the chain length and suggests an adjacent reentry chain-folded lamellar structure incorporating the ap β-sheet architecture. A structure with polar ap β-sheets and γ-turns, stacking with the hydrophobic methyl groups of the alanyl residues in contact, is selected by X-ray structure refinement to give the best match with the experimental data. The pattern of crystallization behavior of the poly(AG) x EG family is consistent with the folding periodicity being in-phase with the amino acid sequence so that the glutamic acid residues are confined to the lamellar surfaces.
The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca2+ spiking in ...the Medicago truncatula root epidermis.
Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca2+ spiking responses and LC-MS to characterize targeted molecules in GSEs.
This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca2+ spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca2+ spiking in M. truncatula ROCs.
We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization.
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Metabolic Syndrome (MetS) is a cluster of cardiovascular (CV) risk factors associated with functional limitations and an increased risk of CV mortality. Resting diastolic function is ...thought to be altered in MetS and may contribute to increased CV risk and functional deficiency. Using echocardiography we explored the relationship between resting diastolic function and left ventricular (LV) contractile reserve capacity, assessed by the change from rest to peak exercise in end‐systolic elastance (ΔEes), in Mets (n=21) and healthy controls (n=13). MetS had resting diastolic dysfunction and a 15% decrease in ΔEes (p<0.05) vs. controls. Univariate analysis, used to examine the correlation between ΔEes and resting diastolic function revealed that ΔEes was related to LV end‐diastolic pressure (EDP), the transmitral to mitral annular early diastolic velocity ratio (E/e’), the diastolic stiffness constant (β), and the isovolumetric relaxation time constant (τ) (p<0.05). We used multivariate analysis to examine the relationship between ΔEes and diastolic function after adjusting for age and metabolic risk score (MRS: the sum of risk points selected for specific CV risk factors). After adjusting for age and MRS, ΔEes remained associated with LV EDP, E/e’, β, and τ (p<0.05), suggesting a link between resting diastolic dysfunction and altered CV reserve in metabolic disease states. AHA 11CRP7370056, 5T32HL090610–04.
Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular ...mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.
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