Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells

AbstractPlasmids containing the origin of replication, oriP, of the Epstein-Barr virus (EBV) and EBV nuclear antigen-1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species-specific replication of EBV, but also the possibility that an EBV-based vector can be used for gene transfer experiments in non-primate cells and an animal experiment regarding human gene therapy

A trial of somatic gene targeting in vivo with an adenovirus vector

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ(+ )gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means

BNIP3 Plays Crucial Roles in the Differentiation and Maintenance of Epidermal Keratinocytes

Transcriptome analysis of the epidermis of Hes1−/− mouse revealed the direct relationship between Hes1 (hairy and enhancer of split-1) and BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), a potent inducer of autophagy. Keratinocyte differentiation is going along with activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process. We found that BNIP3 was expressed in the suprabasal layer of the epidermis, where autophagosome formation is normally observed. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEKs) resulted in autophagy induction and keratinocyte differentiation, whereas knockdown of BNIP3 had the opposite effect. Intriguingly, addition of an autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that BNIP3 plays a crucial role in keratinocyte differentiation by inducing autophagy. Furthermore, the number of dead cells increased in the human epidermal equivalent of BNIP3 knockdown keratinocytes, which suggests that BNIP3 is important for maintenance of skin epidermis. Interestingly, although UVB irradiation stimulated BNIP3 expression and cleavage of caspase3, suppression of UVB-induced BNIP3 expression led to further increase in cleaved caspase3 levels. This suggests that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data provide valuable insights into the role of BNIP3 in the differentiation and maintenance of epidermal keratinocytes

Quantitative Estimate of CO2 Emission Reduction from Reuse of Automobile Parts in Japan

In general, reusing automobile parts reduces not only the cost of replacing the failed parts but also the environmental load of manufacturing new parts. However, these effects have not yet been quantified. The present study focuses on determining the emitted CO2 during production and quantitatively evaluating its reduction by the reuse of automobile parts. First, CO2 emissions are calculated during the reused parts production process at the factory site. Thirty-nine automobiles from 27 models prepared in Japan are examined to measure the amount of CO2 emitted in the production of new parts. Furthermore, the CO2 emission reduction effect for different automobile models is estimated through multiple regression analysis. The CO2 emissions are assumed to be the objective variable, whereas the explanatory variables are derived from the data provided in the automobile inspection certificates. The presented quantitative estimate of CO2 emission reduction owing to the exploitation of reused parts is expected to promote policies for further reducing CO2 emissions and arouse public awareness regarding the benefits of recycling automobile parts

744-3 Inhibition of Nitric Oxide Synthesis does not Increase Cardiac Contractile Response but Reduces Coronary Blood Flow Response to β-Adrenergic Stimulation in Normal Dogs

Although the induction of nitric oxide (NO) synthesis has been implicated as a cause of cytokine-induced depression of cardiac β-adrenergic responsiveness. whether the NO system constitutively present in the normal myocardium plays a role in its physiologic response to β-adrenergic stimulation in vivoremains controversial. Accordingly, we examined the effects of low and high doses of NW-nitro-L-arginine methyl ester (L-NAME)(10 and 100 μg/kg/min for 10 min), an NO synthase inhibitor, administered into left circumflex coronary artery (LCX) on responses of peak left ventricular (LV) dP/dt, regional wall thickening in LCX region and LCX blood flow to graded intracoronary (IC) doses of isoproterenol (ISO:0.002 to 0.016 μg/kg/min) in 7 anesthetized dogs. IC L-NAME was associated with dose-related reductions in IC acetylcholine-induced coronary vasodilation. Effects of L-NAME on ISO-induced changes are shown:baselineISO:0.0020.0040.008.0016Peak LV dP/dt (mmHg/sec) (n=7)control2029±1362586±1922820±2003309±2554120±419*low L-NAME2171±1492566±1762894±2063214±2233707±250*high L-NAME2114±1662326±1932560±1523014±1403354±171*Wall thickening (%) (n=2)control22±725±629±533±735±9low L-NAME25±1125±1528±1931±1836±21high L-NAME28±1725±1525±1531±1934±15LCX blood flow (ml/min)(n=7)control33±648±752±661±870±9*low L-NAME36±741±844±947±852±9*high L-NAME33±736±838±740±748±8*mean ± SEM*p<0.05Thus, inhibition of NO synthesis by L-NAME did not change baseline contractility nor did it increase its response to ISO. It also did not alter baseline blood flow, but reduced significantly its response to ISO. These data strongly suggest that the NO system in the normal myocardium does not modulate contractility, but NO formation in the vasculature contributes to the β-adrenergic coronary vasodilation

Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System

Objective: Human adipose-derived mesenchymal stromal/stem cells (hASC) constitute an attractive source of stemcells for cell-based therapies in regenerative medicine and tissue engineering as they are easy to acquire fromlipoaspirate, expansion, and genetic modification ex vivo. The combination of Pdx-1, MafA, and NeuroD1 has beenindicated to possess the ability to reprogram various types of cells into insulin-producing cells. The aim of this study is toinvestigate whether MafA and NeuroD1 would cooperate with Pdx-1 in the differentiation of hASC into insulin-producingcells.Materials and Methods:In this experimental study, we generated polycistronic expression vectors expressing Pdx1and MafA/NeuroD1 with a reporter from a human EF-1α promoter using 2A peptides in a single tet-off lentiviral vectorsystem. Briefly, hASC were transduced with the lentiviral vectors and allowed to differentiate into insulin-producing cellsin vitro and in vivo. Thereafter, RNA expression, dithizone staining, and immunofluorescent analysis were conducted.Results: Cleaved transcriptional factors from a single tet-off lentiviral vector were functionally equivalent to their nativeproteins and strictly regulated by doxycycline (Dox). Insulin gene expression in hASC transduced with Pdx1, Pdx1/MafA, and Pdx1/NeuroD1 in differentiation medium were successfully increased by 1.89 ± 0.39, 4.81 ± 0.98, 5.51 ±0.63, respectively, compared to venus-transduced, control hASC. These cells could form dithizone-positive cell clustersin vitro and were found to express insulin in vivo.Conclusion: Using our single tet-off lentiviral vector system, Pdx-1 and MafA/NeuroD1 could be simultaneouslyexpressed in the absence of Dox. Further, this system allowed the differentiation of hASC into insulin-producing cells

Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on Human Laminin 111-Coated Dishes

SummaryThe establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy

A study on ensuring the quality and safety of pharmaceuticals and medical devices derived from processing of autologous human induced pluripotent stem(-like) cells

As a series of endeavors to establish suitable measures for the sound development of regenerative medicine using human stem cell-based products, we studied scientific principles, concepts, and basic technical elements to ensure the quality and safety of therapeutic products derived from autologous human iPS cells or iPS cell-like cells, taking into consideration scientific and technological advances, ethics, regulatory rationale, and international trends in human stem cell-derived products. This led to the development of the Japanese official Notification No. 0907-4, “Guideline on Ensuring the Quality and Safety of Pharmaceuticals and Medical Devices Derived from the Processing of Autologous Human Induced Pluripotent Stem(-Like) Cells, ” issued by Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labour and Welfare of Japan, on September 7, 2012. The present paper addresses various aspects of products derived from autologous human iPS cells (or iPS cell-like cells), in addition to similar points to consider that are described previously for autologous human stem cell-based products. Major additional points include (1) possible existence of autologous human iPS cell-like cells that are different from iPS cells in terms of specific biological features; (2) the use of autologous human iPS(-like) cells as appropriate starting materials for regenerative medicine, where necessary and significant; (3) establishment of autologous human iPS(-like) cell lines and their characterization; (4) cell banking and/or possible establishment of intermediate cell lines derived from autologous human iPS(-like) cells at appropriate stage(s) of a manufacturing process, if necessary; and (5) concerns about the presence of undifferentiated cells in the final product; such cells may cause ectopic tissue formation and/or tumorigenesis. The ultimate goal of this guidance is to provide suitable medical opportunities as soon as possible to the patients with severe diseases that are difficult to treat with conventional modalities

Environmental Load Evaluation of Reuse Parts for Automobiles

Abstract Reuse parts are parts removed from scrap automobiles that can be still used. In general, reuse parts reduce not only the cost for replacement of failed parts but also the environmental load. This study quantitatively evaluates environmental loads, such as the amount of CO2 emission during the production of brand new parts, in order to quantify the beneficial effect of the reuse parts. The amount of CO2 emission can be calculated from the power consumption and operating time of each tool and machine employed. Reuse parts generate 0.62 kg of CO2 per automobile when produced, which corresponds to 1,212 kg per year. However, the amount of CO2 emitted from scrapping automobiles without producing new replacement parts is 3,063 kg per year. Therefore, the production of replacement parts emits three times less CO2 than scrapping