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.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline is a sedative regulator of acute stress in the brain of neonatal chicks

The purpose of the present study was to clarify the central nervous system function of amino acids during acute stress. In Experiment 1, changes in free amino acid pattern were investigated in the brain of neonatal chicks exposed to either restraint with isolation-induced or fasting stress. l-Proline and l-arginine were decreased in the telencephalon and diencephalon under any stress. Since the central nervous system functions of l-arginine during the stress response has recently been reported, in Experiment 2, the effect of intracerebroventricular injection of l-proline (0.5, 1.0, 2.0 μmol) during isolation-induced stress was investigated. l-Proline induced sedative and hypnotic effects in a dose-dependent manner. It is suggested that l-proline may have an important role to attenuate the stress response in the central nervous system of chicks.     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

The Effect of Inbreeding on Mating Behaviour of West Indian Sweet Potato Weevil Euscepes postfasciatus

When there is an inbreeding depression, mating with a kin individual is generally considered maladaptive behaviour. However, in some conditions, the inclusive fitness benefits from inbreeding may outweigh the costs of inbreeding depression, and thus, inbreeding tolerance is often adaptive. Inbreeding depression and the effect of relatedness on mating behaviour in the West Indian sweet potato weevil Euscepes postfasciatus were examined. No significant inbreeding depression was detected as indicated by body weight and number of progeny emerging from sweet potato roots. Male mating performance (i.e. number of mating occurrences per night) was adversely affected by inbreeding depression, but the effect was low (fitness loss was 6.3%). Although there were no significant differences in latency to mounting, pre‐copulatory guarding, copulation and post‐copulatory guarding duration between full‐sib and non‐kin pairs, the copulation rate of full‐sib pairs was significantly higher than that of non‐kin pairs. These results support the theoretical prediction that when inbreeding depression is weak, copulation with close relative individuals is favoured.     

NCYM,a Cis-Antisense Gene of MYCN,Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.     

Ischemia-Related Subcellular Redistribution of Sodium Channels Enhances the Proarrhythmic Effect of Class I Antiarrhythmic Drugs: A Simulation Study

Background

Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na⁺) channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na⁺ channels in the arrhythmogenicity under ischemia remain unclear.

Methods

Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na⁺ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.

Results

We found in the myofiber model that the subcellular redistribution of the Na⁺ channels under myocardial ischemia, decreasing in Na⁺ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism) did not reproduce the conduction slowing caused by the subcellular Na⁺ channel redistribution. Furthermore, Na⁺ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na⁺ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.

Conclusions

Proarrhythmia of Na⁺ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na⁺ channel redistribution.     

Diabetic Osteopenia by Decreased β-Catenin Signaling Is Partly Induced by Epigenetic Derepression of sFRP-4 Gene

In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.