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101.
Michael K. Conway Michael J. Gerger Erin E. Balay Rachel O'Connell Seth Hanson Neil J. Daily Tetsuro Wakatsuki 《Journal of visualized experiments : JoVE》2015,(99)
Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications. 相似文献
102.
Koichi Fujisawa Shuji Terai Toshihiko Matsumoto Taro Takami Naoki Yamamoto Hiroshi Nishina Makoto Furutani-Seiki Isao Sakaida 《PloS one》2015,10(6)
Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging. 相似文献
103.
Nobukatsu Horita Kiichiro Tsuchiya Ryohei Hayashi Keita Fukushima Shuji Hibiya Masayoshi Fukuda Yoshihito Kano Tomohiro Mizutani Yasuhiro Nemoto Shiro Yui Ryuichi Okamoto Tetsuya Nakamura Mamoru Watanabe 《Biochemical and biophysical research communications》2014
Background and aims
The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.Methods
Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.Results
We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.Conclusions
The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus. 相似文献104.
Dayanidhi Sarkar Masahiro Yabusaki Yuta Hasebe Pei Yee Ho Shuji Kohmoto Takayuki Kaga Kazuyuki Shimizu 《Applied microbiology and biotechnology》2010,87(1):127-136
The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h−1. The 13C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When
glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate
increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO2 production rate and the biomass concentration decreased, where the 13C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less
active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became
negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate
was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate
concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate
concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase
and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as
the feed gluconate concentration was increased. 相似文献
105.
Chizuru Akatsu Duriya Fongmoon Shuji Mizumoto Jean-Claude Jacquinet Prachya Kongtawelert Shuhei Yamada Kazuyuki Sugahara 《Glycoconjugate journal》2010,27(4):387-399
Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage
region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the
roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans,
and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated
tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of
the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase
or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished
the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based
on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it
was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained
wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required
for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the
linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study
the linkage region are lacking. 相似文献
106.
Yasuhiro Ohshima Mitsutoshi Tsukimoto Takato Takenouchi Hitoshi Harada Akina Suzuki Mitsuru Sato Hiroshi Kitani Shuji Kojima 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.Methods
Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X7 receptor.Results
Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X7 receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X7 receptor channel plays a significant role in mediating the ATP release.General Significance
We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation. 相似文献107.
108.
Jürgen Geisler Takashi Suzuki Hildegunn Helle Yasuhiro Miki Shuji Nagasaki Nhat K. Duong Dagfinn Ekse Turid Aas Dean B. Evans Per E. Lønning Hironobu Sasano 《The Journal of steroid biochemistry and molecular biology》2010,118(4-5):237-241
Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E2; estrone, E1; estrone sulfate, E1S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E2 levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E1 and E1S levels was observed in 677+ breast cancers. In contrast, tumor levels of E2 were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E2 (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E1 (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E1S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E2 were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis. 相似文献
109.
Takashi Kobayashi Taro Ehara Takanori Mizuoka Shuji Adachi 《Biotechnology letters》2010,32(11):1679-1684
In order to synthesize a sugar ester at high concentration, 1,2-O-isopropylidene-α-d-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified
with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value
(240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1,200 mmol palmitic acid/kg solvent were used
in this solvent mixture. 相似文献
110.
Tatsutoshi Inuzuka Hironori Suzuki Masato Kawasaki Hideki Shibata Soichi Wakatsuki Masatoshi Maki 《BMC structural biology》2010,10(1):25