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Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells. Using the newly developed STREAMING-tag system, the authors find that clusters of RNA polymerase II and BRD4 are formed specifically in the transcriptionally active state near the Nanog gene in mouse embryonic stem cells. [ABSTRACT FROM AUTHOR]

Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies. Synopsis: Cholesterol is highly abundant in primary cilia membranes and regulates signal transduction in this specialized organelle. Peroxisomes deliver intracellular cholesterol to the ciliary membrane, a process that is defective in peroxisome‐deficient ciliopathies Peroxisomes move along microtubules to access the ciliary pocket.Rabin8/Rab10/KIFC3 complex mediates peroxisome translocation to primary cilia.ORP3 controls cholesterol transport at the peroxisome–cilium contact sites.Cholesterol supplementation rescues impaired Shh signaling in the cilia of cells from the Zellweger syndrome patients. [ABSTRACT FROM AUTHOR]

The in vivo mechanisms in chronic myocarditis remain unclear. The aim of the current study was to clarify the genomic difference of amyocarditic (CB3O) and myocarditic (CB3M) coxsackievirus B3 (CB3) and the pathogenesis of in vivo mechanisms in chronic myocarditis. We examined the histopathology of CB3-inoculated wild-type (WT) and severe combined immunodeficient (SCID) mice with and without adoptive transfer of lymphocytes. There were no differences in viral growth between CB3O and CB3M. There were four to six nucleotide differences in the sequence of CB3O in comparison with the known CB3M. The difference in virus sequence between CB3O and CB3M was very minimal. The changes were located in 1A, 1C, and 1D regions, which encode the structural capsid proteins. Definite myocarditis developed in WT CH (H-2) inoculated with CB3M. On the contrary, trivial or mild myocarditis occurred in WT CH mice inoculated with CB3O. In SCID CH and SCID CBL/6 (H-2) mice, definite myocarditis developed by inoculation with both CB3O and CB3M. Myocardial lesion was less severe in the mice infected with CB3O than in those with CB3M. After anti-CD8 antibody treatment, myocarditis was easily induced in mice originally showing resistance to infection. In addition, chronic myocarditis developed in CB3O-infected SCID CH mice reconstituted with CB3M-sensitized splenocytes of WT CH mice. The development of chronic myocarditis primarily depends on the presence or absence of the virus genome, and secondarily on the complex interaction between virus virulence and immunological background of the host. CB3 infection may cause chronic myocarditis with ongoing inflammation with or without viral persistence. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) are self-renewing and capable of differentiating into any of the three germ layers. An interesting feature of mESCs is the presence of cell-to-cell heterogeneity in gene expression that may be responsible for cell fate decisions. Nanog, a key transcription factor for pluripotency, displays heterogeneous expression in mESCs, via mechanisms that are not fully understood. To understand this variability, we quantitatively analyzed Nanog transcription and found that Nanog was both infrequently transcribed, and transcribed in a pulsatile and stochastic manner. It is possible that such stochastic transcriptional activation could contribute to the heterogeneity observed in Nanog expression as ''intrinsic noise.'' To discriminate the effects of both intrinsic noise from other (extrinsic) noise on the expression variability of Nanog mRNA, we performed allele-specific single-molecule RNA fluorescent in situ hybridization in a reporter cell line and found that intrinsic noise contributed to approximately 45% of the total variability in Nanog expression. Furthermore, we found that Nanog mRNA and protein levels were well correlated in individual cells. These results suggest that stochastic promoter activation significantly affects the Nanog expression variability in mESCs. [ABSTRACT FROM AUTHOR]

Sulfatases such as arylsulfatase and heparan sulfate 6- O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis. [ABSTRACT FROM AUTHOR]

Charge-transfer (CT) complexation between indoxyl sulfate (3-indolyl hydrogen sulfate) (INS[sup-]: electron donor) and 9,10-anthraquinone-2-sulfonate (2-AQS[sup-]: electron acceptor) around cationic polymers (poly(allylammonium chloride) (PAAH[sup+]Cl[sup-]) and poly(1, 1-dimethylpiperidinium-3,5-diylmethylene chloride) (PDPD[sup+]Cl[sup-])) were studied by absorption and fluorescence spectroscopies and molecular orbital calculation. New broad UV bands corresponding to CT complexation between InS[sup-] and 2-AQS[sup-] at low concentration (∼ 10[sup-4] M) was observed when these polymers were present in the systems. A continuous variation method for the InS[sup-]/2-AQS[sup-]/polymer systems showed that a novel type (INS[sup-]: 2-AQS[sup-] = 2:1) CT complex preferentially formed in the vicinity of these polyions. Stoichiometric ratio of the present CT complex was not affected by the structure and charge density of polyelectrolytes. [ABSTRACT FROM AUTHOR]

We investigated the effects of dibutyryl cyclic adenosine 3′,5′-monophosphate (BtcAMP) on the differentiation of Dunn osteosarcoma cells. Flow-cytometric analysis and DNA synthesis assay showed that BtcAMP decreased the cell population in the S phase in a time- and dose-dependent manner. Also, the cells showed distinct morphological and functional alterations: the cell morphology changed to a fibroblast-like appearance with long and thin protoplasmic processes, the knobs or blebs on both the cell membrane and nuclear membrane disappeared and the intracellular alkaline phosphatase activity increased. Moreover, BtcAMP-treated cells secreted a large quantity of fibronectin, which was deposited on the extended cell surface in the culture medium. Thus, Dunn osteosarcoma cells are differentiated morphologically and functionally by BtcAMP, and might be transformed to benign precursor cells. [ABSTRACT FROM AUTHOR]

We investigated the effect of β-cyclodextrin-benzaldehyde (CDBA) on lymphokine-activated killer (LAK) cell activity of spleen cells from normal or RCT(+)H-2-sarcoma-bearing C3H/He mice. CDBA augmented the induction of LAK cytotoxicity in vitro against RCT(+)H-2 tumor cells by IL-2, whereas the culture with CDBA alone did not. In a LAK cytotoxicity assay in vitro, the augmentative effect of CDBA was strongly exerted against spleen cells originating from 2-week-tumor-bearing mice, rather than those from normal mice or mice that had born tumors for 5 weeks. Such an augmentative effect was not observed against other tumor cells (YAC-1, D-6, Colon-26 and EL-4 cells) non-specifically. When the intravenous adoptive transfer of LAK cells was carried out in the mice, LAK cells from tumor-bearing mice induced by combined culture with interleukin-2 (IL-2) and CDBA markedly inhibited the pulmonary metastases of RCT(+)H-2 tumor, while neither LAK cells from the same tumor-bearing mice induced by only IL-2 nor those from normal mice inhibited the pulmonary metastasis. The majority of LAK cells induced either by IL-2 plus CDBA or by IL-2 alone were found to be Thy1.2 and asialoGM1 cells by flow-cytometric analysis, but no obvious phenotypical difference was observed between them. However, the most significant effect of CDBA might be the maintenance of the Lyt-2 cell level in the spleen cells from tumor-bearing mice. These results suggested that the costimulation of spleen cells with IL-2 and CDBA might induce cytotoxic T cells specific for syngeneic tumor cells. [ABSTRACT FROM AUTHOR]

We have established low- and high-metastatic clones, named RCT(-) and RCT(+) cells, respectively, from the RCT (Radiological, Chiba, and Toyama) sarcoma spontaneously developed in a C3H/He male mouse by the limiting-dilution method in vitro or by the combination of the lung passages and limiting-dilution methods. After 20 serial passages in vitro, the metastatic potential of each clone did not alter. Morphologically, both cells were spindle-shaped, but RCT(+) cells were slightly thicker and larger than RCT(-) cells. The organization of actin-containing filaments was slightly poorer in RCT(+) cells than that in RCT(-) cells. Marked differences were observed in their growth characteristics and adhesiveness to plastic or collagen-coated surfaces, that is, RCT(+) cells grew more slowly but could adhere more rapidly and firmly to the surfaces than RCT(-) cells. RCT(+) cells were agglutinated by all lectins used but several lectins could not agglutinate RCT(-) cells. These results could be a reflection of the difference in oligosaccharide residues on the surface of each cell and, in part, might reflect the difference in organization of the actin-containing filaments that regulate the mobility of lectin receptors. No significant difference between these cell clones was noted in their sensitivity to natural-killer-cell-mediated cytotoxicity in vitro. RCT(-) and RCT(+) cells are considered to be the most useful experimental model for the study of the certain sarcomas. [ABSTRACT FROM AUTHOR]

Murine RCT(+) sarcoma cells were sorted using a fluorescence-activated cell sorter with regard to the expression of H-2 antigens and then an increased H-2-expressing subclone was established, and named RCT(+)H-2. The experimental metastasis of RCT(+) cells was compared with that of RCT(+)H-2 cells by counting pulmonary colonies on the 21st day after i.v. inoculation of tumor cells (5-10×10/mouse). When mice were inoculated with RCT(+) cells, mean numbers of pulmonary colonies were 2.1(range 0-6), 2.8(range 0-7) using 5×10 and 1×10 cells, respectively. On the other hand, in the mice inoculated with RCT(+)H-2 cells, figures obtained were 7.0(range 4-16), 31.9(range 13-79), using 5×10 and 1×10 cells, respectively. The survival rate of RCT(+)H-2 cells was higher than that of RCT(+) cells, when this was assayed in the early stage after i.v. injection of Cr-labeled cells (1×10 cells/mouse). In addition, RCT(+)H-2 cells were more resistant than RCT(+) cells to lysis mediated by natural killer cells. These data suggest that an increase in metastatic ability was paralleled by an increase in the H-2 antigen expression and a decrease in sensitivity to the natural killer cells. [ABSTRACT FROM AUTHOR]

The effect of β-cyclodextrin-benzaldehyde (CDBA) on experimental pulmonary metastasis in C3H/He mice was examined. In an in vitro assay, the growth of RCT(+) cells was inhibited by 1200 μg/ml CDBA using unrenewed media, and by 600 μg/ml CDBA in that using daily renewed media. When mice were treated daily with CDBA, 3 weeks later the number of lung nodules developing after i.v. injection of 1×10 RCT(+) cells was significantly decreased in a dose-dependent manner, i.e., 73.8%, 85.6%, and 95.7% inhibition was observed following 0.5, 5, and 25 mg CDBA/mouse per day p.o. administration, respectively. The same mice showed almost as much natural killer (NK) activity as normal mice. Therefore, experiments were designed to evaluate the effect of CDBA on the NK activity of tumor-free mice whose immunity had been suppressed by 5-fluorouracil (5FU). Injections of 5FU only suppressed this activity to about 50% of normal mice, but the combined treatment with CDBA negated the suppressive effect of 5FU on NK activity. The results suggested that the inhibition of experimental pulmonary metastasis might be induced by the possible combined effects of CDBA; that is, the direct inhibition of tumors and the augmentation of NK cell activity. [ABSTRACT FROM AUTHOR]

T cell activation is assumed to play a crucial role in many viral infections. An important marker for the activation of T cells is the interleukin-2 receptor (IL-2R); resting T lymphocytes do not bear detectable amounts of IL-2R. AMT13, a rat monoclonal antibody against mouse IL-2R, inhibits interleukin-2-dependent cell growth both in vitro and in vivo. Therefore, to clarify the effects of anti-IL-2R antibody treatment upon coxsackievirus B3 (CB3)-infected C3H/He mice, AMT13, 1 µg/mouse per day, was administered, subcutaneously, starting on day 0 (group 2) in experiment I or on day 7 (group 4) in Experiment II for 7 days, respectively. Groups 1 and 3 were examined as infected controls. In both experiments, there was no significant difference in mortality or in the severity of myocarditis between the treated and the untreated groups. Also, myocardial CB3 titers on day 7 did not differ significantly between groups 1 and 2. In addition, the distribution of activated T cell subsets in the inflamed myocardium was not changed by the treatment, and the paucity of myocardial IL-2R-positive cells was confirmed in all groups. Effects of the antibody treatment were confirmed by a decrease in delayed type hypersensitivity. Although some reports have shown that anti-IL-2R antibody has been successfully applied to ameliorate acute renal graft-versus-host disease, to enhance survival of skin allografts, and to suppress diabetic insulitis, it did not exert a beneficial effect on acute CB3 myocarditis in mice. [ABSTRACT FROM AUTHOR]

To test the therapeutic efficacy of levamisole, 5-week-old DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units of encephalomyocarditis virus. Levamisole (2.5mg/kg/per day) was administered intraperitoneally daily, starting simultaneously with the virus inoculation, in experiment I for 14 days, and daily on days 14 to 28 in experiment II in mice that survived to 14 days after virus inoculation. In experiment I, survival was higher, the severity of myocarditis was less, and myocardial virus titers were lower in treated than in untreated animals. In experiment II, levamisole was not effective. No significant changes in serum neutralizing antibody titers occurred in either experiment. Furthermore, levamisole prevented associated lymphoid organ atrophy induced by the virus infection. An additional in vitro study revealed the absence of anti-viral activity of the drug. Thus, levamisole may have favorable effects upon encephalomyocarditis virus myocarditis by preventing the virus-induced lymphoid organ atrophy and reducing myocardial virus replication in the acute stage. [ABSTRACT FROM AUTHOR]

The aim of this study is to test the therapeutic efficacy of immunomodulation with lobenzarit disodium (CCA) upon coxsackievirus B3 (CB3) myocarditis. Two-week-old CH/He mice were inoculated with 10 plaque-forming units of CB3. CCA, 2.5 mg/kg per day, was administered subcutaneously daily on days 0-14 (Experiment I; group 2) and days 14-28 (Experiment II; group 4). Both treated groups were compared to infected controls (groups 1 and 3). For the analysis of splenic lymphocyte subsets, additional mice in untreated and treated groups were killed on day 7, and the percentages of Thy 1.2 (CD3), L3T4 (CD4) and, Ly 2 (CD8) subsets were analyzed by laser flow cytometry (Experiment III). In Experiment I, the survival rate did not differ significantly between groups 1 and 2. Cellular infiltration in the CCA group was less severe. Myocardial virus titers and serum neutralizing antibody titers did not differ significantly between the two groups. In Experiment II, the survival rates between the two groups did not differ significantly. Myocardial necrosis in the CCA group was less severe compared to the control. In Experiment III, the percentages of Thy 1.2 (CD3) and L3T4 subsets (CD4) of the treated group were significantly higher than those of the control group. Thus, CCA increased splenic T cells and improved cardiac pathology in acute murine CB3 myocarditis. [ABSTRACT FROM AUTHOR]

We studied the appearance of intracardiac mural thrombi with time and the relationship between thrombosis and congestive heart failure (CHF) in murine coxsackievirus B3 (CB3) myocarditis. Fourto six-week-old CH/He mice were inoculated intraperitoneally with CB3 and were observed for 90 days. Mice were sacrificed periodically on days 4, 8, 14, 30, and 90. Among 129 mice with myocarditis, 35 (27.1%) developed CHF and 40 (31.0%) demonstrated thrombi after day 8. The total incidence of thrombosis was significantly higher in mice with CHF (71.4%; 25/35) than in those without CHF (16.0%; 15/94) ( P < 0.001). The present study suggests that CB3 myocarditis carries a significant risk of thromboembolism, and that CHF is a risk factor for the appearance of thrombi. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem-cell transplantation (HSCT) for chronic granulomatous disease (CGD) with a reduced-intensity conditioning regimen can be expected to lead to less therapy-related mortality and late-onset impairment, whereas it has also been reported to increase the risk of unsustained mixed donor chimerism and late rejection after transplantation. Herein, we report a 4-year-old boy with CGD who was successfully treated with unrelated bone marrow transplantation with a reduced-intensity conditioning regimen (RIC). Fludarabine-based RIC, 4 Gy of total body irradiation, 120 mg/kg of cyclophosphamide, and 125 mg/m(2) of fludarabine, was adopted for transplantation, followed with 8.9 x 10(8)/kg mononucleated donor cells infused without T-cell depletion. Although hematopoietic engraftment was rapidly obtained by day +17, he developed unstable donor chimerism. After tacrolimus withdrawal, the patient showed grade III acute graft-versus-host disease (GVHD), and subsequently reached full donor chimerism by day +61. Twelve months post-transplant, the patient has remained well with stable and durable engraftment, 100% donor chimerism, and normal superoxide production, without the requirement of donor lymphocyte infusions (DLI). [ABSTRACT FROM AUTHOR]