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Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-lived rodents with a maximum life span exceeding 37 years. They exhibit a delayed aging phenotype and resistance to age-related functional decline/diseases. Specifically, they do not display increased mortality with age, maintain several physiological functions until nearly the end of their lifetime, and rarely develop cancer and Alzheimer's disease. NMRs live in a hypoxic environment in underground colonies in East Africa and are highly tolerant of hypoxia. These unique characteristics of NMRs have attracted considerable interest from zoological and biomedical researchers. This review summarizes previous studies of the ecology, hypoxia tolerance, longevity/delayed aging, and cancer resistance of NMRs and discusses possible mechanisms contributing to their healthy aging. In addition, we discuss current issues and future perspectives to fully elucidate the mechanisms underlying delayed aging and resistance to age-related diseases in NMRs.

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2. The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2, that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.

Certain mammalian species are resistant to cancer, and a better understanding of how this cancer resistance arises could provide valuable insights for basic cancer research. Recent technological innovations in molecular biology have allowed the study of cancer-resistant mammals, despite the fact that they are not the classical model animals, which are easily studied using genetic approaches. Naked mole-rats (NMRs; Heterocephalus glaber) are the longest-lived rodent, with a maximum lifespan of more than 37 years, and almost never show spontaneous carcinogenesis. NMRs are currently attracting much attention from aging and cancer researchers, and published studies on NMR have continued to increase over the past decade. Cancer development occurs via multiple steps and involves many biological processes. Recent research on the NMR as a model for cancer resistance suggests that they possess various unique carcinogenesis-resistance mechanisms, including efficient DNA repair pathways, cell-autonomous resistance to transformation, and dampened inflammatory response. Here, we summarize the molecular mechanisms of carcinogenesis resistance in NMR, which have been uncovered over the past two decades, and discuss future perspectives.

Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-living rodent species. A reason for their long lifespan is pronounced cancer resistance. Therefore, researchers believe that NMRs have unknown secrets of cancer resistance and seek to find them. Here, to reveal the secrets, we noticed a retrotransposon, long interspersed nuclear element 1 (L1). L1s can amplify themselves and are considered endogenous oncogenic mutagens. Since the NMR genome contains fewer L1-derived sequences than other mammalian genomes, we reasoned that the retrotransposition activity of L1s in the NMR genome is lower than those in other mammalian genomes. In this study, we successfully cloned an intact L1 from the NMR genome and named it NMR-L1. An L1 retrotransposition assay using the NMR-L1 reporter revealed that NMR-L1 was active retrotransposon, but its activity was lower than that of human and mouse L1s. Despite lower retrotrasposition activity, NMR-L1 was still capable of inducing cell senescence, a tumor-protective system. NMR-L1 required the 3′ untranslated region (UTR) for retrotransposition, suggesting that NMR-L1 is a stringent-type of L1. We also confirmed the 5′ UTR promoter activity of NMR-L1. Finally, we identified the G-quadruplex structure of the 3′ UTR, which modulated the retrotransposition activity of NMR-L1. Taken together, the data indicate that NMR-L1 retrotranspose less efficiently, which may contribute to the cancer resistance of NMRs.

Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell‐based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+THY‐1+) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell‐replacement therapies using hMSCs., Although mesenchymal stem/stromal cells (MSCs) are promising for cell therapy, long‐term culture leads to MSC senescence with reduced stem cell capacity. We showed that FZD5, which is specifically expressed in immature MSCs, can serve as a stemness indicator. Controlling FZD5 signaling may improve MSC quality and the outcomes of cell therapy.

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA

Background The naked mole-rat (NMR) is the longest-lived rodent with a maximum lifespan of more than 37 years and shows a negligible senescence phenotype, suggesting that tissue stem cells of NMRs are highly capable of maintaining homeostasis. However, the properties of NMR tissue stem cells, including neural stem cells (NSCs), are largely unclear. Methods Neural stem/progenitor cells (NS/PCs) were isolated from the subventricular zone of the neonate NMR brain (NMR-NS/PCs) and cultured in neurosphere and adherent culture conditions. Expression of NSC markers and markers of neurons, astrocytes, and oligodendrocytes was analyzed by immunocytochemistry. In adherent culture conditions, the proliferation rate and cell cycle of NMR-NS/PCs were assessed and compared with those of NS/PCs from mice (mouse-NS/PCs). The DNA damage response to γ-irradiation was analyzed by immunocytochemistry and reverse transcription-quantitative PCR. Results NMR-NS/PCs expressed several NSC markers and differentiated into neurons, astrocytes, and oligodendrocytes. NMR-NS/PCs proliferated markedly slower than mouse-NS/PCs, and a higher percentage of NMR-NS/PCs than mouse-NS/PCs was in G0/G1 phase. Notably, upon γ-irradiation, NMR-NS/PCs exhibited a faster initiation of the DNA damage response and were less prone to dying than mouse-NS/PCs. Conclusions NMR-NS/PCs were successfully isolated and cultured. The slow proliferation of NMR-NS/PCs and their resistance to DNA damage may help to prevent stem cell exhaustion in the brain during the long lifespan of NMRs. Our findings provide novel insights into the mechanism underlying delayed aging of NMRs. Further analysis of NMR tissue stem cells may lead to the development of new strategies that can prevent aging in humans.

Funder: Calico Life Sciences, LLC, The naked mole‐rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled ‘Surprisingly long survival of premature conclusions about naked mole‐rat biology’ described 28 ‘myths’ which, those authors claimed, are a ‘perpetuation of beautiful, but falsified, hypotheses’ and impede our understanding of this enigmatic mammal. Here, we re‐examine each of these ‘myths’ based on evidence published in the scientific literature. Following Braude et al., we argue that these ‘myths’ fall into four main categories: (i) ‘myths’ that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) ‘myths’ that are based on incomplete understanding, where more evidence is clearly needed; (iii) ‘myths’ where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; ( iv ) ‘myths’ where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term ‘myth’ is particularly inappropriate when applied to competing, evidence‐based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole‐rat biology and attempt to clarify some of these misconceptions.

Stem cells play essential roles in the development and tissue homeostasis of animals and are closely associated with carcinogenesis and aging. Also, the somatic cell reprogramming process to induced pluripotent stem (iPS) cells shares several characteristics with carcinogenesis. In this chapter, we focus on iPS cells and the reprogramming process of somatic cells in the naked mole-rat (NMR), the longest-living rodent with remarkable cancer resistance capabilities. NMR somatic cells show resistance to reprogramming induction, and generated NMR-iPS cells have a unique tumor-resistant phenotype. This phenotype is regulated by expressional activation of the tumor suppressor ARF gene and loss-of-function mutation in oncogene ERAS. Notably, it was also found that NMR somatic cells undergo senescence when ARF is suppressed during reprogramming, which would contribute to the resistance to both reprogramming and cancer in NMR somatic cells. Further studies on reprogramming resistance in NMR somatic cells and their concomitant tumor resistance in NMR-iPS cells would contribute to a better understanding of both cancer resistance and delayed aging in NMRs. In addition, NMR-iPS cells can be used as a new and important cell source for advancing research concerning several extraordinary physiological characteristics of NMR. Furthermore, study of NMR-iPS cells could lead to the development of safer regenerative therapies in the future.

Stem cells play essential roles in the development and tissue homeostasis of animals and are closely associated with carcinogenesis and aging. Also, the somatic cell reprogramming process to induced pluripotent stem (iPS) cells shares several characteristics with carcinogenesis. In this chapter, we focus on iPS cells and the reprogramming process of somatic cells in the naked mole-rat (NMR), the longest-living rodent with remarkable cancer resistance capabilities. NMR somatic cells show resistance to reprogramming induction, and generated NMR-iPS cells have a unique tumor-resistant phenotype. This phenotype is regulated by expressional activation of the tumor suppressor ARF gene and loss-of-function mutation in oncogene ERAS. Notably, it was also found that NMR somatic cells undergo senescence when ARF is suppressed during reprogramming, which would contribute to the resistance to both reprogramming and cancer in NMR somatic cells. Further studies on reprogramming resistance in NMR somatic cells and their concomitant tumor resistance in NMR-iPS cells would contribute to a better understanding of both cancer resistance and delayed aging in NMRs. In addition, NMR-iPS cells can be used as a new and important cell source for advancing research concerning several extraordinary physiological characteristics of NMR. Furthermore, study of NMR-iPS cells could lead to the development of safer regenerative therapies in the future.

Naked mole-rats (NMRs) are the longest-lived rodents, showing minimal aging phenotypes. An unsolved paradox is that NMRs exhibit low intracellular anti-oxidant defence despite minimal aging. Here, we explained a link between these “contradicting” features by a phenomenon termed “senescent cell death (SCD)”—Senescence induced cell death in NMR cells due to their inherent vulnerability to reactive oxygen species and unique metabolic system. In NMR skin, we observed few senescent cells during aging or after ultraviolet irradiation, suggesting suppression of senescent cell accumulation in NMR tissue. We discovered that senescent NMR-fibroblasts induce SCD through retinoblastoma protein activation accompanied by autophagy dysregulation, increased oxidative damage and accelerated H2O2-releasing metabolic pathways. During senescence, NMR cells showed resistance to metabolic remodelling unlike mice. Our findings provide mechanistic insights into how extraordinary aging resistance is accomplished in NMR. This will contribute to the development of senolytic drugs to regulate age-related diseases.

The naked mole-rat (NMR) is a poikilothermic mammal that forms eusocial colonies consisting of one breeding queen, several breeding kings, and subordinates. Despite their poikilothermic feature, NMRs possess brown adipose tissue (BAT), which in homeothermic mammals induces thermogenesis in cold environments. However, NMR-BAT thermogenic potential is controversial, and its physiological roles are unknown. Here, we show that NMR-BAT has beta-3 adrenergic receptor (ADRB3)-dependent thermogenic potential, which contributes to thermogenesis in the isolated queen in non-cold environments. NMR-BAT expressed several brown adipocyte marker genes and showed noradrenaline-dependent thermogenic activity in vitro and in vivo. Although our ADRB3 inhibition experiments revealed that NMR-BAT thermogenesis slightly delays the decrease in body temperature in a cold environment, it was insufficient to maintain the body temperatures of the NMRs. In a non-cold environment, NMRs are known to increase their body temperature by a heat-sharing behavior. Interestingly, we found that the body temperatures of NMRs isolated from the colony were also significantly higher than the ambient temperature. We also show that queens, but not subordinates, induce BAT thermogenesis in isolated, non-cold conditions. Our research provides novel insights into the role and mechanism of thermoregulation in this unique poikilothermic mammal.

Naked mole rats (NMRs) have extraordinarily long lifespans and anti-tumorigenic capability. Recent studies of humans and mice have shown that many age-related diseases, including cancer, are strongly correlated with immunity, and macrophages play particularly important roles in immune regulation. Therefore, NMR macrophages may contribute to their unique phenotypes. However, studies of the roles of macrophages are limited by material restrictions and the lack of an established experimental strategy. In this study, we developed a flow cytometric strategy to identify NMR macrophages. The NMR macrophages were extractable using an off-the-shelf anti-CD11b antibody, M1/70, and forward/side scatter data obtained by flow cytometry. NMR macrophages proliferated in response to human/mouse recombinant M-CSF and engulfed Escherichia coli particles. Interestingly, the majority of NMR macrophages exhibited co-staining with an anti-NK1.1 antibody, PK136. NK1.1 antigen crosslinking with PK136 results in mouse NK cell stimulation; similarly, NMR macrophages proliferated in response to NK1.1 antibody treatment. Furthermore, we successfully established an NMR macrophage cell line, NPM1, by transduction of Simian virus 40 early region that proliferated indefinitely without cytokines and retained its phagocytotic capacity. The NPM1 would contribute to further studies on the immunity of NMRs.

The naked mole-rat (NMR) is the world's longest-living rodent, with a maximum lifespan of longer than 31 years without any evidence of neoplastic disease. Recently, the NMR has come to be regarded as a useful animal model for the study of longevity and cancer resistance. Sequencing analysis of the NMR genome revealed the existence of species-specific changes in the predicted sequence of the INK4a and ARF tumor suppressors, suggesting the possibility that these two genes might have important roles in NMR's unique longevity and cancer resistance. Here, we report the molecular cloning and characterization of the INK4a and ARF genes in vitro. To investigate the expression and function of the INK4a and ARF genes in NMR, we generated several research tools, including antibodies, real-time PCR primer sets, and overexpression and knockdown vectors. Our results showed that endogenous expression of INK4a and ARF was upregulated in NMR fibroblasts treated with DNA-damaging agents or after serial passaging. In addition, overexpression of INK4a or ARF caused cell cycle arrest in both NMR fibroblasts and mouse NIH-3T3 cells. These results suggest INK4a and ARF execute a conserved function as cell cycle inhibitors in NMR. The research tools developed in the present study will be useful for exploring the specific function of INK4a and ARF genes in the unique longevity and cancer-resistant phenotype of NMRs.

Serotonergic axons extend diffuse projections throughout various brain areas, and serotonergic system disruption causes neuropsychiatric diseases. Loss of the cytoplasmic region of protocadherin-α (Pcdh-α) family proteins, products of the diverse clustered Pcdh genes, causes unbalanced distributions (densification and sparsification) of serotonergic axons in various target regions. However, which Pcdh-α member(s) are responsible for the phenotype is unknown. Here we demonstrated that Pcdh-αC2 (αC2), a Pcdh-α isoform, was highly expressed in serotonergic neurons, and was required for normal diffusion in single-axon-level analyses of serotonergic axons. The loss of αC2 from serotonergic neurons, but not from their target brain regions, led to unbalanced distributions of serotonergic axons. Our results suggest that αC2 expressed in serotonergic neurons is required for serotonergic axon diffusion in various brain areas. The αC2 extracellular domain displays homophilic binding activity, suggesting that its homophilic interaction between serotonergic axons regulates axonal density via αC2′s cytoplasmic domain.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR., The naked mole-rat exhibits an exceptional resistance to cancer. Here, the authors show that induced pluripotent stem cells derived from the naked mole-rat lack teratoma-forming tumorigenicity due to a naked mole-rat-specific ARF-dependent tumour-suppression mechanism.

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17′, 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

Hypertension is a common complication after kidney transplantation and adversely affects graft and patient survival. Strategies for anti-hypertensive therapy in an allograft from a hypertensive donor with arteriolosclerosis and the target blood pressure have not been clearly defined. Here, we report the case of deteriorating transplanted kidney function after anti-hypertensive treatment. A 40-yr-old man had received a kidney transplant from a living relative donor (his 69-yr-old father), who was hypertensive and had severe arteriolosclerosis. The recipient showed good allograft function immediately (s-Cr 1.8 mg/dL); however, blood pressure and proteinuria increased markedly two wk after transplantation (blood pressure 180/90, urinary protein 3.4 g/d). We then started anti-hypertensive agents (a calcium channel blocker and an angiotensin II receptor blocker). Blood pressure and proteinuria were corrected to the normal range within two wk of starting the treatment (blood pressure 130/80, urinary protein 0.3 g/d). However, his kidney function continued to deteriorate (s-Cr 2.7 mg/dL). A biopsy, performed at that time, revealed glomerular collapse, advanced interstitial fibrosis and severe arteriolosclerosis, with no evidence of rejection. These findings could have been the result of renal allograft hypoperfusion. We then reduced the anti-hypertensive agents on day 45 after transplantation and observed improved allograft function within a week (s-Cr 1.6 mg/dL). This case suggests that renal hemodynamic responses may be impaired and renal perfusion may not be appropriately maintained in an allograft with severe arteriolosclerosis at a blood pressure level suitable for the recipient. Anti-hypertensive treatment should be performed carefully when the allograft is from an elderly hypertensive donor.

The protocadherin-alpha (Pcdh-alpha) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-alpha genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-alpha gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-alpha isoforms and found that 1) the transcription of specific Pcdh-alpha isoforms correlated significantly with the methylation state of the promoter and the 5' (but not the 3') region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-alpha isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-alpha isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5'-regions of the Pcdh-alphaC1 and alphaC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-alpha1 to -alpha12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5'-region of the first exon may help regulate differential Pcdh-alpha transcription and that hypermethylation is sufficient to repress transcription.

これまでにわれわれは，白血球系細胞除去療法のうちG-CAPとL-CAPが同様に潰瘍性大腸炎（Ulcerative colitis：UC）の末梢血液中の樹状細胞（dendritic cell：DC）Subset比率に影響を与えることを報告した1)．今回，UCで増加する病因細胞をスクリーニングするため，UC症例と健常人で末梢血樹状細胞のサブセット解析を施行した．総樹状細胞（Total DCs）は末梢血有核細胞の4 color FACS解析によりLineage marker（CD3, CD14, CD16, CD19, CD20 and CD56）陰性，かつHLA-DR陽性の細胞として同定され，このTotal DCsはさらにCD11CとCD123の反応性によりDC1，DC2，less differentiated DC：ldDCの各DCサブセットに分類された．末梢血液中のFACS解析の結果，DC SubsetのうちldDCについては吸着療法施行前のUC患者では健常者に比較して有意に増加していた（p＜0.03）．次にldDCがUCの活動性調節細胞である可能性を検討するため，DC細胞数に影響を与える免疫抑制剤（グルココルチコイド）投与量を一定（30±10mg/body）に保ったうえで白血球系細胞吸着療法（L-CAP 6例，G-CAP 6例）を施行したUC症例12例（男性3例，女性9例）を対象に，各DC Subsetの経時変化を検討した．12例中，瀬尾activity index 50点以上低下が得られた白血球系細胞吸着療法の有効例は8例，残りの4例は無効であった．有効例では治療経過とともにldDCは徐々に減少し，最終回には有意な低下が認められ，健常者と同等レベルまで正常化した．一方，吸着療法の無効例では，ldDCは持続高値のままで有意な変化を認めなかった．一方，DC Subsetのうち総DC数，DC1，DC2はUC患者と健常人の比較で有意差を認めず，吸着療法前後で有意な変化を認めなかった．以上から潰瘍性大腸炎で増加する末梢血液中のldDCは，免疫抑制剤の投与量変更なしで白血球系細胞吸着療法により臨床症状とともに正常化することから，UCの活動性調節細胞である可能性が示された．

Two novel compounds, stachyflin and acetylstachyflin, have been isolated by solid-state fermentation of Stachybotrys sp. RF-7260. The structures of both metabolites, determined by detailed NMR analyses and X-ray crystallographic analysis, are novel with a pentacyclic moiety including cis-fused decalin. The absolute stereochemistry of stachyflins was determined by circular dichroism analysis. Stachyflin showed antiviral activity against influenza A virus (H1N1) in vitro with an IC50 value of 0.003 microM. Acetylstachyflin was about 77-fold less active than stachyflin.

In the course of screening for yeast squalene synthase inhibitors, bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260. The related compounds bisabosquals B, C and D were also isolated from Stachybotrys ruwenzoriensis RF-6853. Bisabosquals inhibited squalene synthases. IC50 values of bisabosqual A against the microsomal squalene synthases from Saccharomyces cerevisiae, Candida albicans, HepG2 cell and rat liver were 0.43, 0.25, 0.95 and 2.5 microg/ml, respectively. Bisabosqual C exhibited inhibitory activities similar to bisabosqual A. Bisabosqual A showed broad spectrum antifungal activity in vitro.

The squalene synthase inhibitor bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260, and its structure was determined on the basis of spectroscopic methods including detailed 2D NMR analyses. The structures of bisabosquals B, C and D isolated from Stachybotrys ruwenzoriensis RF-6853 were determined by spectroscopic methods and chemical reactions. The absolute stereochemistry of bisabosquals A, B and D was determined by X-ray crystallographic analysis. They have novel cis-fused tetracyclic structures with a bisabolane-type sesquiterpene and phenol moieties.

Thielocins A2 alpha, A2 beta, A3, B1, B2 and B3 were isolated as a novel family of phospholipase A2 inhibitors from the fermentation broth of Thielavia terricola RF-143 together with thielavins and thielocins A1 alpha and A1 beta. The most potent inhibitory activity (IC50 = 0.0033 microM) against rat group II phospholipase A2 was shown by thielocin A1 beta. Against human group II phospholipase A2, thielocin B3 (IC50 = 0.076 microM) was the most potent.

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.

Pluripotent stem cells can provide us with an enormous cell source for in vitro model systems for development. In 2006, new methodology was designed to generate pluripotent stem cells directly from somatic cells, and these cells were named induced pluripotent stem cells (iPSCs). This method consists of technically simple procedures: donor cell preparation, gene transduction, and isolation of embryonic stem cell-like colonies. The iPSC technology enables cell biologists not only to obtain pluripotent stem cells easily but also to study the reprogramming events themselves. Here, we describe the protocols to generate iPSCs from somatic origins by using conventional viral vectors. Specifically, we state the usage of three mammalian species: mouse, common marmoset, and human. As mouse iPSC donors, fibroblasts are easily prepared, while mesenchymal stem cells are expected to give rise to highly reprogrammed iPSCs efficiently. Common marmoset (Callithrix jacchus), a nonhuman primate, represents an alternative model to the usual laboratory animals. Finally, patient-specific human iPSCs give us an opportunity to examine the pathology and mechanisms of dysregulated genomic imprinting. The iPSC technology will serve as a valuable method for studying genomic imprinting, and conversely, the insights from these studies will offer valuable criteria to assess the potential of iPSCs.

Cinatrins A, B, C1, C2 and C3, a family of phospholipase A2 inhibitors were isolated from the fermentation broth of Circinotrichum falcatisporum RF-641. They were found to be novel spiro-gamma-dilactones and gamma-lactones derived from 1,2,3,5-tetra or 1,2,3(or 1,2,4)-trihydroxypentadecane-1,2,3-tricarboxylic acids. Structures were elucidated by MS and NMR studies and chemical transformations. The structure of cinatrin C3 was confirmed by X-ray crystallographic analysis, and its absolute configuration was determined by comparison of the CD spectra with related compounds.

A novel compound depudecin inducing the flat phenotype of ras- and src- transformed NIH3T3 cells at a concentration of 1 μg/ml was isolated from the culture broth of Alternaria brassicicola. Based on its spectroscopic characteristics and X-ray crystallographic analysis of its bis-(1S)-(-)-camphanate, the structure of depudecin was determined'to be (2R, 3S, 4S, 5E, lS, 8S, 9R)-2, 9-dihydroxy3, 4;7, 8-diepoxy-undeca-5, 10-diene.

The clustered protocadherin-alpha (Pcdha) genes, which are expressed in the vertebrate brain, encode diverse membrane proteins whose functions are involved in axonal projection and in learning and memory. The Pcdha cluster consists of 14 tandemly arranged genes (Pcdha1-Pcdha12, Pcdhac1, and Pcdhac2, from 5' to 3'). Each first exon (the variable exons) is transcribed from its own promoter, and spliced to the constant exons, which are common to all the Pcdha genes. Cerebellar Purkinje cells show dual expression patterns for Pcdha. In individual Purkinje cells, different sets of the 5' genes in the cluster, Pcdha1-12, are randomly expressed, whereas both 3' genes, Pcdhac1 and Pcdhac2, are expressed constitutively. To elucidate the relationship between the genomic structure of the Pcdha cluster and their expression in Purkinje cells, we deleted or duplicated multiple variable exons and analyzed the expression of Pcdha genes in the mouse brain. In all mutant mice, transcript levels of the constant exons and the dual expression patterns were maintained. In the deletion mutants, the missing genes were flexibly compensated by the remaining variable exons. On the other hand, in duplication mutants, the levels of the duplicated genes were trimmed. These results indicate that the Pcdha genes are comprehensively regulated as a cluster unit, and that the regulators that randomly and constitutively drive Pcdha gene expression are intact in the deleted or duplicated mutant alleles. These dual regulatory mechanisms may play important roles in the diversity and fundamental functions of neurons.

BACKGROUND Encapsulating peritoneal sclerosis (EPS) is a serious complication in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) or automated peritoneal dialysis (APD). The aim of this study was to find a predictor for EPS. METHODS Patients with EPS who were detected by a historical cohort study using clinical data of 219 CAPD patients at our hospital. We recruited 25 patients with EPS who were compared with the patients without EPS who were matched for age and dialysis period as controls. Differences between the two groups (non-EPS group and EPS group) with respect to age, gender, primary disease, dialysis period, serum urea nitrogen, serum creatinine, beta2MG, CRP and PET (peritoneal equilibration test) category (determined by the peritoneal function testing) were analyzed. RESULTS According to multiple regression analysis, a high beta2MG level was an independent risk factor for EPS (odds ratio 1.162, 95% confidence interval 1.026 - 1.317, p = 0.018). Other clinical markers did not show positive significance. A ROC (receiver operating characteristic) curve was prepared to evaluate the suitability of I(2)2MG measurement as a screening test. The sensitivity was 64% and the specificity was 80% when a beta2MG level of 37.0 mg/dl was taken as the cut-off value. The odds ratio for occurrence of EPS was 8.8 when beta2MG level was in the range of 35 - 40 mg/dl, 13.5 when I(2)2MG level was > 40 mg/dl and 1 when beta2MG level was < 30 mg/dl. CONCLUSION These findings suggest that beta2MG is useful as a screening test for the onset of EPS, and that beta2MG and accumulation of middle-molecular uremic substances may be related to the pathophysiology of EPS.

PA48009, a lanthionine-containing peptide antibiotic was isolated from the culture broth of Streptoverticillium griseoverticillatum PA-48009, and identified as duramycin. Determination of the structure using both Edman degradations and 2D NMR spectroscopy showed the need to revise the structure of duramycin given in literature. Duramycin (PA48009) was different from lanthiopeptin (Ro 09-0198, cinnamycin) only by a Lys/Arg exchange at position 2.

New cyclotetrapeptides, trapoxins A and B were isolated from the culture broth of Helicoma ambiens RF-1023. These compounds exhibit detransformation activities against v-sis oncogene-transformed NIH3T3 cells (sis/NIH3T3) as antitumor agents. The structures were found to be new cyclotetrapeptides, cyclo[(S)-phenylalanyl-(S)-phenylalanyl-(R)-pipecolinyl- (2S,9S)-2-amino-8-oxo-9,10-epoxydecanoyl-] for trapoxin A and cyclo[(S)-phenylalanyl-(S)-phenylalanyl-(R)-prolyl-2- amino-8-oxo-9,10-epoxydecanoyl-] for trapoxin B, by X-ray analysis, mass spectrometric, NMR and chemical studies.

This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell. This protocol can be completed in 5-6 d.

The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-alpha and Pcdh-gamma gene clusters are expressed in all of the vertebrates studied. For the Pcdh-alpha isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation. Here we investigated total allelic gene regulation in the Pcdh-alpha and -gamma clusters, including the C-type variable exons (C1 to C5) and the Pcdh-gammaA and -gammaB variable exons in single Purkinje cells. Using split single-cell reverse transcription-PCR analysis, almost all of the Purkinje cells at postnatal day 21 biallelically expressed all the C-type isoforms, whereas the Pcdh-alpha isoforms showed both monoallelic and combinatorial expression. The Pcdh-gammaA and -gammaB isoforms also showed differential regulation in each cell with both monoallelic and combinatorial gene regulation. These data indicated that different types of allelic gene regulation (monoallelic versus biallelic) occurred in the Pcdh-alpha and -gamma clusters, although they were spliced into the same constant exons. It has been reported that each C-type Pcdh-alpha or -gamma transcript has a different expression pattern during brain development, suggesting that the different C-type variable exons may code temporal diversity, although the Pcdh-alpha, -gammaA, and -gammaB isoforms were differential and combinatorial gene regulation within a single cell. Thus, the multiple gene regulations in the Pcdh-alpha and -gamma clusters had a potential mechanism for increasing the diversity of individual neurons in the brain.

Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.

The kidney deeply takes part in phosphorus and vitamin D metabolism. The chronic kidney disease patients usually suffer from serious bone disease. They are prescribed the glucocorticoid medication for primary kidney disease, as nephrotic syndrome and collagen renal disease, with aging. The end of therapeutic target is normal bone structure with normal bone turnover in CKD patients. The control of serum Ca and phosphorus level is important step and also another target. This issue describes the problem and the advancement of treatment and the measurement of the bone mass from this viewpoint.

New inhibitors of aldose reductase, designated salfredins A3, A4, A7, C1, C2, C3 and B11, were isolated from the fermentation broth of Crucibulum sp. RF-3817 by successive purification procedures of solvent extraction, silica gel column chromatographies and reverse-phase HPLC. Their structures were established by spectroseopic methods, including UV, SI-MS and NMR. The structures of salfredins A4 and B11 were confirmed by X-ray crystallographic analysis.

Background Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by the forced expression of defined transcription factors. Although most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear. Methodology/Principal Findings Here, we generated iPS cells from PDGFRα+ Sca-1+ (PαS) adult mouse mesenchymal stem cells (MSCs) and PDGFRα− Sca-1− osteo-progenitors (OP cells), and compared the induction efficiency and quality of individual iPS clones. MSCs had a higher reprogramming efficiency compared with OP cells and Tail Tip Fibroblasts (TTFs). The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency. Conclusions/Significance Our findings suggest that a purified source of undifferentiated cells from adult tissue can produce high-quality iPS cells. In this context, prospectively enriched MSCs are a promising candidate for the efficient generation of high-quality iPS cells.

The FcγRllla (FCGR3A) 158V/V allotype displays a higher antibody-dependent cellular cytotoxicity compared to the FCGR3A-158V/F or 158F/F allotype, leading to susceptibility to rheumatoid arthritis, better clinical response to rituximab in NHL, and lower risk of periodontitis. We tested the hypothesis that FCGR3A polymorphism influences the development of GVHD and GVL, and TRM. The FCGR3A-158V/F genotype was determined in 49 recipient and donor pairs who underwent HLA-matched SCT in our institute. Subjects with the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were almost one-third and twice as likely to develop chronic GVHD respectively (Table 1). The recipients with the FCGR3A-158V/V allotype had a 60% increase in probability of 3-year TRM. Multivariate analysis showed that the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were independent risk factors for chronic GVHD (HR = 0.8 and 1.2, respectively), as well as the unrelated donor BMT (HR = 1.6) and the related donor PBSCT (HR = 2.4). The recipient FCGR3A-158V/V was an independent predictor for 3-year TRM (HR = 4.0) with the high risk disease (HR = 3.0) and the cord blood transplantation (HR = 5.0). We conclude that the recipient and donor FCGR3A polymorphism is associated with the development of chronic GVHD and TRM after HLA-matched SCT. These findings could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic SCT. Besides, these raise the question of whether the genetic risk factor in a recipient might also be involved in the mechanisms of chronic GVHD. Table 1 | FCGR3A allotype | 2–4 acute GVHD(HR) | Chronic GVHD (HR) | 3 YR TRM (HR) | |:---------------------------------:| ------------------ | ----------------- | ------------- | | *P

New antibiotics, PA-46101 A and B, were isolated from the culture broth of a Streptomycete. The molecular formulae of A and B were determined to be C52H70O18 and C61H86O22, -respectively, by elemental analyses, NMR and mass spectrometry. Their structures were elucidated by X-ray crystallography and NMR spectroscopy. These antibiotics are active in vitro against anaerobic Gram-positive and Gram-negative bacteria and also against a limited number of aerobic Gram-positive bacteria.