Abbreviations: HPLC, high performance liquid chromatography; MS, mass spectrum; PCR, polymerase chain reaction; PLR, pinoresinol lariciresinol reductase; SDG, secoisolariciresinol diglucoside; Ubi, ubiquitin. were found to be 2.2-times higher in one of the three positive transgenic sub-lines at TB 2 B than that in the wild-type (117.9 ± 4.5 vs. 52.9 ± 19.8 µg/g, p < 0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.
Immunoglobulin (Ig) domains are the most prevalent protein domain structure and share a highly conserved folding pattern; however, this structural family of proteins is also the most diverse in terms of biological roles and tissue expression. Ig domains vary significantly in amino acid sequence but share a highly conserved tryptophan in the hydrophobic core of this beta-stranded protein. Palladin is an actin binding and bundling protein that has five Ig domains and plays an important role in normal cell adhesion and motility. Mutation of the core tryptophan in one Ig domain of palladin has been identified in a pancreatic cancer cell line, suggesting a crucial role for this sole tryptophan in palladin Ig domain structure, stability, and function. We found that actin binding and bundling was not completely abolished with removal of this tryptophan despite a partially unfolded structure and significantly reduced stability of the mutant Ig domain as shown by circular dichroism investigations. In addition, this mutant palladin domain displays a tryptophan-like fluorescence attributed to an anomalous tyrosine emission at 341 nm. Our results indicate that this emission originates from a tyrosinate that may be formed in the excited ground state by proton transfer to a nearby aspartic acid residue. Furthermore, this study emphasizes the importance of tryptophan in protein structural stability and illustrates how tyrosinate emission contributions may be overlooked during the interpretation of the fluorescence properties of proteins.
The cytoskeleton, composed of actin, microtubules, and associated motor and binding proteins, self-organizes into different structures and morphologies to drive diverse mechanical processes in eukaryotic cells. While in vitro actomyosin networks are well-characterized, few studies have examined how the structure and activity of these networks are altered in the presence of microtubules. We previously synthesized steady-state actin-microtubule networks, and found that mechanical properties, such as network elasticity, depend on molecular interactions between actin and microtubules, such as degree of crosslinking and filament bundling. Here, we create active actinmicrotubule networks by adding the motor protein myosin. Using confocal microscopy and microrheology, we characterize how the network structure evolves during motor activity, and connect how microscopic changes in network structure affect the mechanical properties of the network at the mesoscopic scale. Our results shed important new light on how actinmicrotubule interactions influence the structure, mechanics and activity that motor-driven cytoskeleton networks exhibit.
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