Production of Rat Diploid Parthenogenones by Suppression of 2nd Polar Body Extrusion and their Preimplantation Development

幼若ラット過排卵卵を用いて,排卵卵母細胞をヒアルロニダーゼ・低温処理により,第2極体放出を抑制することによって,2倍体胚へ発育しうる活性卵を作出することを目的とし,さらにそれらの着床前における発育性を卵管in-vivo培養に続くin-vitro培養によって検討した. 卵令20-23時間卵において卵をヒアルロニダーゼ30-60分処理後5℃又は8℃のいずれかの低温にさらすことにより高率に第2極体放出抑制活性卵が得られ,そのほとんどは,"2前核"卵であった. 次に第2極体放出抑制活性卵作出に及ぼす卵令の影響について検討した結果,最適卵令20-23時間の結果とは異なり,卵令18時間では,活性化は誘起されなかった. 卵令26-27時間卵では,fragmentation卵と多核を有した卵が非常に多く観察された. この結果より卵令は活性化誘起に対し,Critical factorであることが示された. 本実験で得られた. "第2極体+1前核"卵は,2細胞期あたりで発育を停止したが,しかし,"2前核"卵は,桑実胚あるいは胚盤胞へ発育することがわかった. また,得られた単為発生胚盤胞の倍数性は40の染色体を持つ2倍体と判定された. 以上の結果より,ラット単為発生胚の着床前発育にとって,2倍体胚の重要性が示された

Studies on the Induction of Parthenogenetic Mammalian Embryos : In vitro Activation of Superovulatory Eggs of Immature Rat

幼若ラット過排卵卵をヒアルロニダーゼ・低張処理法により活性化させるための条件を検討し,その活性化法を確立することを目的とした. さらに,得られた活性卵の発育性を体外培養で観察し,活性卵の正常性について,形態的観察からも検索した. 卵令20～21時間卵の場合,高い活性率を得るためのヒアルロニダーゼ(100mits/ml)処理条件と低張処理条件は,それぞれ90分と3/5Mまたは,3.5/5Mを組み合わせた場合であり,低張処理において,等張液から低張媒液へ除々にでなく直ちに卵を処理することが必要であった. 卵令25～26時間卵の活性条件は,ヒアルロニダーゼ90分処理のみで,活性化し,低張処理を必要としなかった. 一方,卵令16時間卵は,ヒァルロニダーゼ処理90分,低張処理3/5Mあるいは3.5/5Mの活性条件では,活性卵は得られず,さらに,ヒァルロニダーゼ処理120分,低張処理3/5Mと活性刺激を強めてもほとんど活性卵は得られなかった. これらのことより,卵令に伴う第2次成熟分裂紡錘糸の変化(軸転そして卵中央部への移動)が,卵の活性化に影響を及ぼしていることが推察された. どの処理条件においても,得られた活性卵の発育型は,ほとんど“第2極体十1前核”であり,それらは,半数体と推定された. 次に,これらの活性卵の発育性を体外培養で観察した結果,それらの40～60%が第1分割分裂期を経て,受精卵の場合とほぼ同じ時間経過で2-cellへ発育した. しかし,2-cell以後の発育は,ほとんど観察されなかった

Ca2+-free Medium Mediated Parthenogenetic Activation of Mouse Oocytes

1.Ca2+_free媒地による活性化誘起によってICR系マウスではHCG後令20時間を越えた卵母細胞からは50%以上の活性化卵が得られ,そのほとんどが2前核か1前核の2倍体卵子であった. 2.ICR系の誘起排卵卵子では50%以上の活性化率が得られたが,自然排卵卵子では活性化されなかった. さらに幼若動物からの卵子に若干の活性化率の低下が認められたが,活性化typeにそれらと成熟ドナーからの卵子との間にはほとんど差は認められなかった. 又,22時間令の卵において4CS系統で80%以上の高い活性化率がみられ,4CS系統に2PB+1PNの半数体活性卵が多いことを除いては,ICR,RFおよび4CS系のドナーの系統による差異は認められなかった. 3.活性化のためのCa2+_free媒地での処理時間が再検討され,処理時間が15分に短縮されても活性化率は低下しなかったが,2PB+1PNの半数体卵子が増加することから,2倍体卵子を得るためには,少くとも1時間のCa2+_free処理が必要である. 4.IonophoreA23187によってCa2+の存否にかかわらず高率に2倍体活性化卵子が得られたことから,マウス卵細胞の活性化にCa2+の関与が強く示唆された

Peripheral Administration of Morphine Attenuates Postincisional Pain by Regulating Macrophage Polarization through COX-2-Dependent Pathway

BACKGROUND: Macrophage infiltration to inflammatory sites promotes wound repair and may be involved in pain hypersensitivity after surgical incision. We recently reported that the development of hyperalgesia during chronic inflammation is regulated by macrophage polarity, often referred to as proinflammatory (M1) or anti-inflammatory (M2) macrophages. Although opioids such as morphine are known to alter the inflammatory milieu of incisional wounds through interactions with immunocytes, the macrophage-mediated effects of morphine on the development of postincisional pain have not been well investigated. In this study, we examined how morphine alters pain hypersensitivity through phenotypic shifts in local macrophages during the course of incision-induced inflammation. RESULTS: Local administration of morphine in the early phase, but not in the late phase alleviated mechanical hyperalgesia, and this effect was reversed by clodronate-induced peripheral depletion of local macrophages. At the morphine-injected incisional sites, the number of pro-inflammatory F4/80(+)iNOS(+)M1 macrophages was decreased during the course of pain development whereas increased infiltration of wound healing F4/80(+)CD206(+)M2 macrophages was observed during the early phase. Morphine increased the gene expression of endogenous opioid, proenkephalin, and decreased the pronociceptive cytokine, interleukin-1β. Heme oxygenase (HO)-1 promotes the differentiation of macrophages to the M2 phenotype. An inhibitor of HO-1, tin protoporphyrin reversed morphine-induced analgesic effects and the changes in macrophage phenotype. However, local expression levels of HO-1 were not altered by morphine. Conversely, cyclooxygenase (COX)-2, primarily produced from peripheral macrophages in acute inflammation states, was up-regulated in the early phase at morphine-injected sites. In addition, the analgesic effects and a phenotype switching of infiltrated macrophages by morphine was reversed by local administration of a COX inhibitor, indomethacin. CONCLUSIONS: Local administration of morphine alleviated the development of postincisional pain, possibly by altering macrophage polarity at the incisional sites. A morphine-induced shift in macrophage phenotype may be mediated by a COX-2-dependent mechanism. Therefore, μ-opioid receptor signaling in macrophages may be a potential therapeutic target during the early phase of postincisional pain development

Elevated expression of CD30 in adult T-cell leukemia cell lines: possible role in constitutive NF-κB activation

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL). HTLV-1 encoded Tax1 oncoprotein activates the transcription of genes involved in cell growth and anti-apoptosis through the NF-κB pathway, and is thought to play a critical role in the pathogenesis of ATL. While Tax1 expression is usually lost or minimal in ATL cells, these cells still show high constitutive NF-κB activity, indicating that genetic or epigenetic changes in ATL cells induce activation independent of Tax1. The aim of this study was to identify the molecules responsible for the constitutive activation of NF-κB in ATL cells using a retroviral functional cloning strategy. RESULTS: Using enhanced green fluorescent protein (EGFP) expression and blasticidin-resistance as selection markers, several retroviral cDNA clones exhibiting constitutive NF-κB activity in Rat-1 cells, including full-length CD30, were obtained from an ATL cell line. Exogenous stable expression of CD30 in Rat-1 cells constitutively activated NF-κB. Elevated expression of CD30 was identified in all ATL lines examined, and primary ATL cells from a small number of patients (8 out of 66 cases). CONCLUSION: Elevated CD30 expression is considered one of the causes of constitutive NF-κB activation in ATL cells, and may be involved in ATL development

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo.

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer

More than the “Wife Corps”: Female Tenant Farmer Struggle in 1920s Japan

AbstractStruggles over social reproduction intensified and took on new forms in Japan during the interwar period, as the state found it increasingly difficult to secure the foundations for the continued accumulation of capital. Landlord-tenant disputes that erupted nationwide in the midst of Japan's post-World War I agricultural recession was one concrete manifestation of these struggles. While the significance of tenant disputes has been analyzed in great detail by scholars, there has been a surprising lack of historical scholarship on the role that female tenant farmers played within them. This absence is a manifestation of two tendencies: First, gendered assumptions surrounding the figure of the tenant farmer have led scholars of agrarian social movements to work from a relatively limited understanding of what constitutes struggle and by extension, who its protagonists have been. Second, the conflation of waged work as productive work and by extension, non-waged work as unproductive has unwittingly relegated many forms of struggle that working women participated in to the realm of the pre-political. This paper contends that far from being mere supporters – the wife corps – of what was ultimately a male-driven movement, female participants in tenant disputes produced their own powerful critiques of the way that the Japanese state and capital undervalued their lives and labor. As such, they should be understood as one link in a rich history of proletarian feminist struggle both within and outside of the Japanese empire