MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.     

Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration.     

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes

In this study, we created porcine‐induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c‐Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA‐Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537–553, 2017. © 2016 Wiley Periodicals, Inc.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Chronotherapy of maxacalcitol on skin inflammation induced by topical 12-O-tetradecanoylphorbol-13-acetate in mice

ABSTRACT

In general, chronotherapy is desirable for a more effective and/or safe dosage regimen. In this study, a daily rhythm of skin vitamin D receptor (VDR) and chronotherapeutic profiles of maxacalcitol, a vitamin D analogue, were evaluated using mice with skin inflammation induced by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This study showed that skin nuclear VDR expression in TPA-treated mice has a daily rhythm with the peak at the middle of active period. The effects of maxacalcitol were greater after dosing during early to middle of active period than those after dosing during early to middle of inactive period. These data suggest that chronotherapeutic profiles of maxacalcitol partly depend on the daily rhythm of skin nuclear VDR in TPA-treated mice. Because TPA-treated mice are considered as one of animal models of psoriasis, these animal data might be helpful for establishing chronotherapeutic approach of maxacalcitol in clinical practice.     

Transposon insertion mutagenesis of a genetic region encoding serum resistance in an 80 kb plasmid of Salmonella dublin

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3.0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 2, while the region for mouse lethality was within a 6.0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9.6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15.0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.     

Incidence of antibodies against human immunodeficiency virus, human T-cell lymphotropic virus type 1, hepatitis B virus, hemorrhagic fever with renal syndrome virus and Chlamydia in Tonga and western Samoa

Among the populations of Tonga and Western Samoa, serum antibodies against human immunodeficiency virus or hemorrhagic fever with renal syndrome virus were not detected (0/904 and 0/192). No serum samples were considered to be positive for antibody against human T-cell lymphotropic virus type 1 (0/527). Hepatitis B antigen and antibody were found in 4% (8/192) and 47% (90/192), respectively. Chlamydia trachomatis IgG and C. psittaci IgG antibodies were detected in 39% (75/192) and 47% (91/192), respectively. The possibilities of the spread of human immunodeficiency virus and hemorrhagic fever with renal syndrome virus on the islands when the viruses invade from abroad were discussed.