Stability of cytochromes c′ from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure

Extremophiles - The stability of dimeric cytochrome c′ from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the...     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.     

Selection responses of means and inbreeding depression for female fecundity in Drosophila melanogaster suggest contributions from intermediate-frequency alleles to quantitative trait variation

The extent to which quantitative trait variability is caused by rare alleles maintained by mutation, versus intermediate-frequency alleles maintained by balancing selection, is an unsolved problem of evolutionary genetics. We describe the results of an experiment to examine the effects of selection on the mean and extent of inbreeding depression for early female fecundity in Drosophila melanogaster. Theory predicts that rare, partially recessive deleterious alleles should cause a much larger change in the effect of inbreeding than in the mean of the outbred population, with the change in inbreeding effect having an opposite sign to the change in mean. The present experiment fails to support this prediction, suggesting that intermediate-frequency alleles contribute substantially to genetic variation in early fecundity.     

A 350-S recovery period does not necessarily allow complete recovery of peak power output during repeated cycling sprints

The aim of this study was to determine whether a 350-s recovery period allows recovery of peak power output (PPO) to its initial value under the condition of a blood lactate (La) concentration higher than 10 mmol.L-1 during repeated cycling sprints (RCS). RCS (10x10-s cycling sprints) were performed under two conditions. Under one condition, the recovery period of RCS was fixed at 35 s (RCS35), and under the other condition, a 350-s recovery period was set before the 5th and 9th sets, and a 35-s recovery period was set before the other sets (RCScomb). In RCScomb, PPO in the 5th set recovered to that in the 1st set, but PPO in the 9th set did not. Under both conditions, blood La concentration progressively increased and reached approximately 14 mmol.L-1 at the end of the RCS. In RCScomb, VO2 immediately before the 5th set was not significantly different from that immediately before the 9th set. Mean power frequency (MPF) values estimated by a surface electromyogram from the vastus lateralis in the 5th and 9th sets were significantly higher in RCScomb than in RCS35. In conclusion, a 350-s recovery period does not allow recovery of PPO to its initial value under the condition of a blood La concentration of 14 mmol.L-1 during RCS.     

The biomechanics of amnion rupture: an X-ray diffraction study

Pre-term birth is the leading cause of perinatal and neonatal mortality, 40% of which are attributed to the pre-term premature rupture of amnion. Rupture of amnion is thought to be associated with a corresponding decrease in the extracellular collagen content and/or increase in collagenase activity. However, there is very little information concerning the detailed organisation of fibrillar collagen in amnion and how this might influence rupture. Here we identify a loss of lattice like arrangement in collagen organisation from areas near to the rupture site, and present a 9% increase in fibril spacing and a 50% decrease in fibrillar organisation using quantitative measurements gained by transmission electron microscopy and the novel application of synchrotron X-ray diffraction. These data provide an accurate insight into the biomechanical process of amnion rupture and highlight X-ray diffraction as a new and powerful tool in our understanding of this process.     

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole-imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection.