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21.
Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells 总被引:4,自引:0,他引:4
Sumimoto H Tsuji T Miyoshi H Hagihara M Takada-Yamazaki R Okamoto S Ikeda Y Takahashi T Kawakami Y 《Journal of immunological methods》2002,271(1-2):153-165
Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC. 相似文献
22.
The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region 总被引:1,自引:0,他引:1
Miyazaki J Jozaki M Nakatani N Watanabe T Saba R Nakada K Hirabayashi T Yonemura I 《Journal of muscle research and cell motility》1999,20(7):655-660
To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced. 相似文献
23.
Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes 总被引:5,自引:0,他引:5
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The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system. 相似文献
24.
Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer 总被引:8,自引:0,他引:8
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Furukawa T Sunamura M Motoi F Matsuno S Horii A 《The American journal of pathology》2003,162(6):1807-1815
We previously found frequent loss of heterozygosity at 12q21 and 12q22-q23.1 in primary pancreatic cancers, and the DUSP6/MKP-3 gene residing in this region at 12q22 lost its expression in the great majority of pancreatic cancer cell lines. The DUSP6/MKP-3 protein is a dual-specificity phosphatase that dephosphorylates the active form of ERK, making a feedback loop to control ERK activity. Gain-of-function mutations of KRAS2 occur in the great majority of pancreatic cancer cells, and loss of expression of DUSP6/MKP-3 may synergistically promote constitutive activation of ERK and uncontrolled cell growth. To study loss of the feedback pathway and its impact on pancreatic cancer cell growth, we first investigated the expression of DUSP6/MKP-3 in primary pancreatic cancer tissues immunohistochemically; we found up-regulation in mildly as well as severely dysplastic/in situ carcinoma cells and down-regulation in invasive carcinoma, especially in the poorly differentiated type. Adenovirus-mediated reintroduction of DUSP6/MKP-3 into cultured pancreatic cancer cells induced strong expression of recombinant DUSP6/MKP-3 and reduction of phosphorylated ERK in a dose-dependent manner based on the multiplicity of infection and resulted in suppression of cell growth. Moreover, analyses by flow cytometry and immunocytochemistry revealed that the exogenous expression of DUSP6/MKP-3 induced apoptosis. These results show that DUSP6 exerts apparent tumor-suppressive effects in vitro and suggest that DUSP6 is a strong candidate tumor suppressor gene at 12q22 locus. 相似文献
25.
PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis 总被引:20,自引:2,他引:20
Kobayashi Kazuhiro; Mlzuno Kunihiko; Hida Akiko; Komakl Rie; Tomita Keiko; Matsushita Ikumi; Namlki Mikio; Iwamoto Teruaki; Tamura Shohzoh; Minowada Shlgeru; Nakahori Yutaka 《Human molecular genetics》1994,3(11):1965-1967
We analyzed DNA from 63 Japanese men with either azoospermiaor severe oligospermia whose Y chromosomes were cytogeneticallynormal. A total of 16 loci were examined: 15 loci on the longarm between DYS7E and DYZ1, and the YRRM1 locus, a candidategene for the azoospermic factor, AZF. One patient with a perlcentricinversion of the Y chromosome was also included. We detectedmicro-deletions in ten individuals. The YRRM1 gene was Involvedin only three of them. The remaining seven patients showed deletionbetween DYS7C and DYS239 in common, indicating the presenceof at least one additional gene, deletion of which causes azoospermia. 相似文献
26.
Kazuhiko Orikasa Shin-ichi Fukushige Senji Hoshi Seiichi Orikasa Keiichi Kondo Yasuhide Miyoshi Yoshinobu Kubota A. Horii 《Journal of human genetics》1998,43(4):228-230
Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular
mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported
in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers.
These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients.
Received: February 6, 1998 / Accepted: July, 3, 1998 相似文献
27.
28.
Gert Baumann Stephan B. Felix Claus D. Heidecke Gotthard Rieß Ursula Loher Liesel Ludwig Prof. Dr Hans Blömer 《Inflammation research》1984,15(3-4):216-228
Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to -adrenergic and H2-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [3H]-dihydroalprenolol ([3H]-DHA), [3H]-methyl-tiotidine ([3H]-TIOT) and [3H]-quinuclidinyl benzilate ([3H]-QNB) to cardiac
1-, H2- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of-sympathomimetic, in contrast to administration of prenalterol and the conventional therapy with -sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventriculardP/dt
max was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (K
D) of the remaining -receptors while specific [3H]-TIOT- and [3H]-QNB-binding was unchanged.Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [3H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal -receptors. Furthermore, treatment with H2-agonists in combination with -blocking agents may have beneficial effects, whereas conventional therapy with -sympathomimetic drugs tends to worsen the already depressed function of the -adrenergic stimulation mechanism.Supported by grants Ba 666/1 and Ba 666/2-2 from the Deutsche Forschungsgemeinschaft (DFG).Data presented in this paper are part of a doctoral thesis by Dr S.B. Felix. 相似文献
29.
Toru Furukawa Rumi Fujisaki Yoshitaro Yoshida Naomi Kanai Makoto Sunamura Tadayoshi Abe Kazunori Takeda Seiki Matsuno Akira Horii 《Modern pathology》2005,18(8):1034-1042
DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms. 相似文献
30.
Association of Epstein-Barr virus with oral cancers 总被引:2,自引:0,他引:2