Engineering Fructosyl Peptide Oxidase to Improve Activity Toward the Fructosyl Hexapeptide Standard for HbA1c Measurement

The recently discovered fructosyl peptide oxidase from Phaeosphaeria nodorum (PnFPOX) was demonstrated to react with the glycated hexapeptide measurement standard of hemoglobin A1c, fVHLTPE. The highly reactive Coniochaeta FPOX (FPOX-C) showed no detectable activity with the hexapeptide. Two loop regions were identified as having important effects on the enzymatic properties of FPOX. The first loop has a strong influence on the ability to bind larger glycated peptides, while the second loop has a significant effect on catalytic activity. Loop-substitution mutants showed that the highest activity against fVHLTPE resulted from the combination of the first loop from PnFPOX and the second loop from FPOX-C. The most promising engineered FPOX created, which showed 17-fold greater dehydrogenase activity against fVHLTPE than wild-type PnFPOX, was the FPOX-C mutant with a PnFPOX-derived loop 1 region and an Asn56Ala substitution.     

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism

Plant Molecular Biology - Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired...     

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP‐170

AMP‐activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP‐170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell–cell junction present between cardiomyocytes. A contractile inhibitor, MYK‐461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK‐461, suggesting that the localization of AMPK is regulated by mechanical stress. Time‐lapse imaging analysis revealed that the inhibition of CLIP‐170 Ser‐311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK‐461 increased the individual cell area of cardiomyocytes in CLIP‐170 phosphorylation‐dependent manner. Moreover, heart‐specific CLIP‐170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP‐170 at the intercalated disks.     

Long-term survival of transplanted induced pluripotent stem cell-derived neurospheres with nerve conduit into sciatic nerve defects in immunosuppressed mice

Since the advent of induced pluripotent stem cells (iPSCs), clinical trials using iPSC-based cell transplantation therapy have been performed in various fields of regenerative medicine. We previously demonstrated that the transplantation of mouse iPSC-derived neurospheres containing neural stem/progenitor cells with bioabsorbable nerve conduits promoted nerve regeneration in the long term in murine sciatic nerve defect models. However, it remains unclear how long the grafted iPSC-derived neurospheres survived and worked after implantation. In this study, the long-term survival of the transplanted mouse iPSC-derived neurospheres with nerve conduits was evaluated in high-immunosuppressed or non-immunosuppressed mice using in vivo imaging for the development of iPSC-based cell therapy for peripheral nerve injury. Complete 5-mm long defects were created in the sciatic nerves of immunosuppressed and non-immunosuppressed mice and reconstructed using nerve conduits coated with iPSC-derived neurospheres labeled with ffLuc. The survival of mouse iPSC-derived neurospheres on nerve conduits was monitored using in vivo imaging. The transplanted iPSC-derived neurospheres with nerve conduits survived for 365 days after transplantation in the immunosuppressed allograft models, but only survived for at least 14 days in non-immunosuppressed allograft models. This is the first study to find the longest survival rate of stem cells with nerve conduits transplanted into the peripheral nerve defects using in vivo imaging and demonstrates the differences in graft survival rate between the immunosuppressed allograft model and immune responsive allograft model. In the future, if iPSC-derived neurospheres are successfully transplanted into peripheral nerve defects with nerve conduits using iPSC stock cells without eliciting an immune response, axonal regeneration will be induced due to the longstanding supportive effect of grafted cells on direct remyelination and/or secretion of trophic factors.     

Strategies for maintaining Na⁺ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogentic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.     

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens.     

Antimalarial activity of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) and its carboxylic acid derivatives

Malaria is one of the world's deadliest diseases and is becoming an increasingly serious problem as malaria parasites develop resistance to most of the antimalarial drugs used today. We previously reported the in vitro and in vivo antimalarial potencies of 1,2,6,7-tetraoxaspiro[7.11]nonadecane (N-89) and 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Plasmodium falciparum and Plasmodium berghei parasites. To improve water-solubility for synthetic peroxides, a variety of cyclic peroxides having carboxyl functionality was prepared based on the antimalarial candidate, N-251, and their antimalarial activities were determined. The reactions of N-89 and its derivatives with Fe(II) demonstrated a highly efficient formation of the corresponding carbon radical which may be suspected as a key for the antiparasitic activity.     

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy

Background aimsTransplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model.MethodsASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10⁵ cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed.ResultsASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group.ConclusionsCompared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.     

hzAnalyzer: detection,quantification, and visualization of contiguous homozygosity in high-density genotyping datasets

The analysis of contiguous homozygosity (runs of homozygous loci) in human genotyping datasets is critical in the search for causal disease variants in monogenic disorders, studies of population history and the identification of targets of natural selection. Here, we report methods for extracting homozygous segments from high-density genotyping datasets, quantifying their local genomic structure, identifying outstanding regions within the genome and visualizing results for comparative analysis between population samples.