Abstract Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) facilitate the engraftment of human (h) hematopoietic stem cells when transplanted simultaneously in animal and human studies. ...However, the type of MSCs that preferentially enhance the engraftment of HSCs is unknown. Recent studies have shown that MSCs derived from a single source are heterogeneous in terms of cell size, morphology, proliferation rate, and differentiation potential. This study was designed to investigate the properties of UCB-MSCs, which influence the engraftment of hHSCs in a NOD/SCID mouse model. We categorized MSCs as being the most effective (UCB-352 MSCs) or the least effective (UCB-156 MSCs) at promoting the homing and engraftment of HSCs, and compared the characteristics of these 2 MSC populations. We observed that the 2 populations showed differences in characteristics typical of immature MSCs, and related to proliferation potential. We showed that UCB-352 MSCs, which proliferate quickly, preferentially enhanced the engraftment of HSCs in NOD/SCID mice. In addition, we observed differences in the pattern of both PODXL and Oct4 expression, and in the levels of cytokines such as SDF-1 and SCF using flow cytometry and membrane arrays. The more effective UCB-352 MSCs expressed higher levels of PODXL and Oct4, which were associated with immaturity, than did the UCB-156 MSCs. Furthermore, UCB-352 cells secreted greater levels of SDF-1 and SCF, both of which are required for hematopoiesis. We propose that the proliferation potential of UCB-MSCs, coupled with their immature characteristics, may serve as a novel standard to promote the homing and engraftment of HSCs.
Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances ...that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal
p-nitrophenol hydroxylase,
p-nitroanisole
O-demethylase and aminopyrine
N-demethylase activities significantly only in male mice. Increases in
Cyp2e1 and
Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.
The discovery of a giant dielectric constant of 105 in CaCu3Ti4O12 has increased interest in this perovskite-type oxide. Here we demonstrate that, in addition to high permittivity, CaCu3Ti4O12 has ...remarkably strong nonlinear current-voltage characteristics without the addition of any dopants. An intrinsic electrostatic barrier at the grain boundaries is responsible for the unusual nonlinear behaviour. The nonlinear coefficient of CaCu3Ti4O12 reaches a value of 900, which is even greater than that of the varistor material ZnO. As a result, CaCu3Ti4O12 may lead to efficient switching and gas-sensing devices.
Dielectric and piezoelectric properties of lead-free (1-x)(Bi
0.5
K
0.5
)TiO
3
-xBiFeO
3
ceramics prepared by a conventional solid-state reaction method were studied in the range of x = 0∼40 mol%. A ...tetragonal perovskite structure was maintained at x = 0∼10 mol% forming a solid solution between (Bi
0.5
K
0.5
)TiO
3
and BiFeO
3
with no secondary phase. Piezoelectric coefficient (d
33
) increased from 31 pC/N at x = 0 mol% to 64 pC/N at x = 6 mol% and then decreased with further addition of x. Depolarization temperature (T
d
) gradually decreased with increasing x from 302°C at x = 0 mol% to 163°C at x = 20 mol% and slightly increased up 180°C at x = 40 mol%.
RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an ...important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.
The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis ...in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.
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