Molecular Evolution of Hemagglutinin (H) Gene in Measles Virus Genotypes D3, D5, D9, and H1

We studied the molecular evolution of H gene in four prevalent Asian genotypes (D3, D5, D9, and H1) of measles virus (MeV). We estimated the evolutionary time scale of the gene by the Bayesian Markov Chain Monte Carlo (MCMC) method. In addition, we predicted the changes in structure of H protein due to selective pressures. The phylogenetic tree showed that the first division of these genotypes occurred around 1931, and further division of each type in the 1960–1970s resulted in four genotypes. The rate of molecular evolution was relatively slow (5.57×10⁻⁴ substitutions per site per year). Only two positively selected sites (F476L and Q575K) were identified in H protein, although these substitutions might not have imparted significant changes to the structure of the protein or the epitopes for phylactic antibodies. The results suggested that the prevalent Asian MeV genotypes were generated over approximately 30–40 years and H protein was well conserved.     

Chromosomal Mapping of Genes in theRBCS Family in Arabidopsis thaliana

In Arabidopsis thaliana, four genes have been identified inthe RBCS gene family, one being assigned to subfamily RBCS-Aand the other three to subfamily RBCS-B (1B, 2B and 3B). Todetermine the chromosomal location ofthese genes, hybridizationanalysis with CIC YAC high-density filters was carried out forthe RBCS-A gene, and CAPS analysis for the three RBCS-B genes,based on the finding that restriction fragment length polymorphismis present in the upstream region of the gene RBCS-3B. The RBCS-Agene was mapped at 100.8 cM from the top of chromosome 1 andthe three RBCS-B genes at 62.70 cM from the top of chromosome5.     

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Sperm Preservation by Freeze-Drying for the Conservation of Wild Animals

Sperm preservation is a useful technique for the maintenance of biological resources in experimental and domestic animals, and in wild animals. A new preservation method has been developed that enables sperm to be stored for a long time in a refrigerator at 4°C. Sperm are freeze-dried in a solution containing 10 mM Tris and 1 mM EDTA. Using this method, liquid nitrogen is not required for the storage and transportation of sperm. We demonstrate that chimpanzee, giraffe, jaguar, weasel and the long-haired rat sperm remain viable after freeze-drying. In all species, pronuclei were formed after the injection of freeze-dried sperm into the mouse oocytes. Although preliminary, these results may be useful for the future establishment of “freeze-drying zoo” to conserve wild animals.     

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

We have developed an oat cell-free apoptosis system to investigate the execution mechanisms of plant apoptosis. Cell extracts derived from oat tissues undergoing toxin (victorin)-induced apoptosis caused nuclear collapse and internucleosomal DNA fragmentation in isolated nuclei. Pharmacological studies revealed that cysteine protease, which is E-64-sensitive but insensitive to caspase-specific inhibitors, is a crucial component in the morphological change of isolated nuclei, and that nuclease and the cysteine protease act cooperatively to induce the apoptotic DNA laddering. Interestingly, this finding is contrasted with those in well-studied animal cell-free systems in which an apoptotic endonuclease is solely responsible for the DNA fragmentation.     

The SNP in the promoter region of the bovine ELOVL5 gene influences economic traits including subcutaneous fat thickness

Genetic analyses have contributed to improvements of economically important traits derived from adipose tissue such as fatty acid composition in beef. Elongation of very long chain fatty acids (ELOVL) genes encode for the enzymes that play an important role in elongation of long-chain fatty acids. In this study, we aimed to discover genetic polymorphisms of ELOVL gene family in cattle populations to develop genetic markers. As a result, five synonymous mutations were detected in the coding regions of the ELOVL1, ELOVL2, ELOVL3 and ELOVL5 genes. In addition, six mutations were identified in promoter region of the ELOVL5. Two of five mutations in the promoter region of ELOVL5 were expected to alter the ELOVL5 expression and influence the economic traits, because of the high synteny of the region which was essential for activation of Elovl5 in mouse. Therefore, we performed association analysis between the genotypes and traits and our result revealed that T allele of g.-110T>C in ELOVL5 gene promoter indicated significantly thinner subcutaneous fat thickness (TT, 2.39 cm; CT, 2.35 cm) than that of C allele (CC, 2.68 cm) in a Japanese Black population. Our results suggest that the g.-110T>C is a useful genetic marker for the breeding in beef cattle.     

Ly6C+Ly6G− Myeloid‐derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C⁺Ly6G^? immature monocyte fraction exhibited the same characteristics as myeloid‐derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C⁺Ly6G^? fraction prior to injury by anti‐Gr‐1 antibody (clone: RB6‐8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo‐generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C⁺Ly6G^? fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC‐based strategy that can be applied to acute inflammatory diseases.