共查询到10条相似文献,搜索用时 140 毫秒
1.
Kenta Nakamura Nathan Salomonis Kiichiro Tomoda Shinya Yamanaka Bruce R. Conklin 《PloS one》2009,4(11)
Background
Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that Gi-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.Methodology/Principal Findings
Specific and irreversible inhibition of Gi-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, Gs-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.Conclusions/Significance
Experiments with pertussis toxin suggest that Gi signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a Gi-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications. 相似文献2.
Minoru Tomizawa Fuminobu Shinozaki Takao Sugiyama Shigenori Yamamoto Makoto Sueishi Takanobu Yoshida 《PloS one》2013,8(8)
Background
Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine.Materials and Methods
Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM.Results
The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells.Conclusion
Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage. 相似文献3.
4.
Nathaniel Edward Bennett Saidu Imad Abu Asali Brigitte Czepukojc Berthold Seitz Claus Jacob Mathias Montenarh 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.Methods
Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H2O2 and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.Results
We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.Conclusions
The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.General Significance
The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds. 相似文献5.
6.
Bicheng Yang Xiaolu Duan Wenzheng Wu Weidong JiWenqi Wu Wen ZhongZhijian Zhao Shujue LiYang Liu Guohua Zeng 《Life sciences》2014
Aim
Promoter-targeted small activating RNAs (saRNAs) have been shown to be able to induce target gene expression, a mechanism known as RNA activation (RNAa). The present study tested whether saRNA can induce the overexpression of TRPV5 in human cells derived from the kidney and subsequently manipulate cell calcium uptake.Main methods
Three saRNAs complementary to the TRPV5 promoter were synthesized and transfected into cells. TRPV5 expression at the RNA and protein levels was analyzed by quantitative real-time PCR and Western blotting respectively. For functional study, transcellular Ca2 + transportation was tested by fura-2 analysis. Dihydrotestosterone (DHT), a suppressor of cellular calcium transportation, was administered to challenge the activating effect of selected saRNA.Key findings
One of these synthesized saRNAs, ds-2939, significantly induced the expression of TRPV5 at both mRNA and protein levels. Fura-2 analysis revealed that the intracellular Ca2 + concentration was elevated by ds-2939. DHT treatment reduced transmembrane Ca2 + transport, which was partially antagonized by ds-2939.Significance
Our results suggest that a saRNA targeting TRPV5 promoter can be utilized to manipulate the transmembrane Ca2 + transport by upregulating the expression of TRPV5 and may serve as an alternative for the treatment of Ca2 + balance-related diseases. 相似文献7.
Hidehiko Akiyama Masahiro Endo Taei Matsui Itsurou Katsuda Nobuhiko Emi Yasuko Kawamoto Takaaki Koike Hidehiko Beppu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011
Background
Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.Methods
Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.Results
Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.Conclusions
These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.General significance
This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients. 相似文献8.
9.
Min-Syuan Huang Tze-Kai Wang Yi-Wen Liu Yi-Ting Li Tang-Hao Chi Chih-Wei Chou Mingli Hsieh 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Carbonic anhydrase 8 (CA8) is an isozyme of α-carbonic anhydrases (CAs). Previous studies showed that CA8 can be detected in human adult brain, with more intense expression in the cerebellum. Single mutations in CA8 were reported to cause novel syndromes like ataxia, mild mental retardation or the predisposition to quadrupedal gait.Methods
In the present study, we examine the functions of CA8 in neuronal cell lines, mouse cerebellar granule neurons and zebrafish.Results and conclusions
We demonstrated that overexpression of CA8 in neuronal cells significantly decreased cell death under staurosporine treatment. Moreover, CA8 overexpression significantly increased cell migration and invasion ability in neuronal cells and in mouse cerebellar granule neurons, implicating that CA8 may be involved in neuron motility and oncogenesis. By using zebrafish as an animal model, motor reflection of 3 dpf zebrafish embryos was significantly affected after the down-regulation of CA8 through ca8 morpholino.Conclusions
We concluded that CA8 overexpression desensitizes neuronal cells to STS induced apoptotic stress and increases cell migration and invasion ability in neuronal cells. In addition, down-regulated CA8 decreases neuron mobility in neuronal cells and leads to abnormal calcium release in cerebellar granule neurons. Knockdown of the ca8 gene results in an abnormal movement pattern in zebrafish.General significance
Our findings provide evidence to support that the impaired protective function of CA8 contributes to human neuropathology, and to suggest that zebrafish can be used as an animal model to study the biological functions of human CA8 in vivo. 相似文献10.
Adriana G. Guimarães Luciana Scotti Marcus Tullius Scotti Francisco J.B. Mendonça Júnior Nayara S.R. Melo Rafael S. Alves Waldecy De Lucca Júnior Daniel P. Bezerra Daniel P. Gelain Lucindo J. Quintans Júnior 《Life sciences》2014