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91.
Genes associated with the formation of germ cells from embryonic stem cells in cultures containing different glucose concentrations 总被引:1,自引:0,他引:1
Mizuno K Tokumasu A Nakamura A Hayashi Y Kojima Y Kohri K Noce T 《Molecular reproduction and development》2006,73(4):437-445
In a previous study, we established a system for visualizing the development of germ cells from mouse embryonic stem (ES) cells in culture using knock-in ES clones in which visual reporter genes were expressed from the mouse vasa homolog, Mvh. While assessing various culture conditions, we found that germ-cell formation was markedly depressed in low glucose medium. Using a repeated polymerase chain reaction (PCR) subtraction method, we identified genes that were differentially expressed in low versus high glucose media. Three genes that were predominantly expressed in high glucose medium, thioredoxin-interacting protein (Txnip), pituitary tumor-transforming gene 1 (Pttg), and RuvB-like protein 2 (RuvBl2), were further investigated. These genes were also found to be highly expressed in adult and embryonic gonads, and RuvBl2 in particular, which encodes an ATP-dependent DNA helicase, was specifically detected in the spermatocytes and spermatids of the adult testis as well as in primordial germ cells. Furthermore, using a green fluorescent protein (GFP) fusion construct, we found that RuvBl2 was expressed in both the nucleus and cytoplasm of testicular germ cells. These findings suggest a possible relationship between glucose metabolism and germ-cell development. 相似文献
92.
Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder. 相似文献
93.
94.
Tsuji AB Sugyo A Sudo H Sagara M Ishikawa A Ohtsuki M Kimura T Ogiu T Miyagishi M Taira K Imai T Harada YN 《Genomics》2006,87(2):236-242
The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat. 相似文献
95.
Yoshida A Matumoto M Hshizume H Oba Y Tomishige T Inagawa H Kohchi C Hino M Ito F Tomoda K Nakajima T Makino K Terada H Hori H Soma G 《Microbes and infection / Institut Pasteur》2006,8(9-10):2484-2491
Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages. 相似文献
96.
Cryopreservation does not alter the allogenicity and development of vasculopathy in post-transplant rat aortas 总被引:2,自引:0,他引:2
BACKGROUND: Cryopreservation is a valuable technique for storing heart valve and vascular allografts. However, the biological ramifications of cryopreservation are still unclear; therefore, using animal experiments we assessed how 'cryopreservation' influences graft allogenicity and cell viability. METHODS: Thoracic aortas of Lewis rats were prepared as fresh (F) or cryopreserved (CP) grafts, and implanted into the infrarenal aorta of Lewis or Brown Norway rats (BNs). The grafts and spleens were harvested at post-operative day 7 and 28 (POD7, POD28) for analyses. RESULTS: First, the systemic immune response to transplantation was estimated by mixed lymphocyte reaction analyses using spleen cells from na?ve or recipient BNs. The alloreactivity of the recipients increased to 1.5 times that of the na?ve BNs at POD7 and POD28, when stimulated by mitomycin C-treated Lewis spleen cells. Second, local immune response was estimated by TNFalpha, IFNgamma, and iNOS mRNA expression in the grafts by quantitative PCR, which revealed 20- to 40-fold increases at POD28 after allotransplantation. Third, endothelial cell viability was estimated by endothelial NOS mRNA expression level: it was similar and highest in F and CP grafts before transplantation then significantly decreased after both syngeneic and allogeneic transplantation. Finally, intimal hyperplasia, expressed by I/M ratio, developed over time after allotransplantation, reaching 2.5 times the thickness of F grafts before transplantation. The results of these experiments revealed no difference between F and CP grafts before and after transplantation. CONCLUSION: Cryopreservation did not modify the allogenicity of vascular allografts and had minimal adverse impacts on graft cell viability. 相似文献
97.
Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms 总被引:2,自引:0,他引:2
Inamatsu M Tochio T Makabe A Endo T Oomizu S Kobayashi E Yoshizato K 《Development, growth & differentiation》2006,48(2):73-86
Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion. 相似文献
98.
Miura A Honma R Togashi T Yanagisawa Y Ito E Imai J Isogai T Goshima N Watanabe S Nomura N 《FEBS letters》2006,580(30):6871-6879
Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1β, tumor necrosis factor-, interferon-β, interferon-γ, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions. 相似文献
99.
We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors. 相似文献
100.
A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended. 相似文献