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981.
A PCR-based protocol for generating West Nile virus replicons 总被引:1,自引:0,他引:1
A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon. 相似文献
982.
It has been difficult cytologically to distinguish salivary duct carcinoma (SDC) from high-grade carcinoma. We investigated the microscopic cytological findings, morphometric image analyses, and immunohistochemical features of SDC, focusing on how we achieved an accurate differential diagnosis distinguishing SDC from salivary gland carcinomas with squamous differentiation. Immunohistochemical staining was performed for androgen receptor (AR), gross cystic disease fluid protein-15 (GCDFP15), mammaglobin, human gastric mucin, MUC1, MUC2, p63, and cytokeratin high molecular weight. Of the 13 cases of SDC, 9 cases showed typical cytological findings of sheet clusters with polygonal granular cytoplasm with fine chromatin. The other 4 cases showed unusual cytological findings of a pseudo-papillary cluster or scattered cells only, and the tumor cells showed coarse chromatin. Morphometric image analysis showed that the nucleus area was statistically different between SDC and salivary gland carcinomas with squamous differentiation. AR-positive expression (P = 0.008), GCDFP15-positive expression (P = 0.005) and p63-negative expression (P = 0.001) were effective as SDC-specific markers in immunohistochemistry. An accurate cytological diagnosis of SDC can be determined by immunostaining with AR, GCDFP15, and p63, based on the nuclear findings. 相似文献
983.
984.
Ito A Hagiyama M Mimura T Matsumoto M Wakayama T Iseki S Yokozaki H Okada M 《Laboratory investigation; a journal of technical methods and pathology》2008,88(5):504-514
Cell adhesion molecule 1 (CADM1), formerly referred to as SgIGSF, TSLC1, or Necl-2, has been characterized as a mast-cell adhesion molecule that mediates efficient interactions with mesothelial cells. Here, we examined whether CADM1 might be involved in the diffuse tumor growth over the pleural surface that characterizes malignant pleural mesothelioma (MPM). Immunohistochemical and western blot analyses revealed that 14 (25%) of 57 MPMs expressed the full-length form of CADM1 on the cell membrane, but non-neoplastic mesothelial cells did not express it at all. The majority of available MPM cell lines also expressed the full-length form of CADM1. We compared CADM1-positive and -negative MPM cells in culture within soft agar and in coculture on mesothelial or fibroblastic monolayers. Within soft agar, CADM1-negative MPM cells were capable of forming colonies, whereas CADM1-positive cells were not, suggesting that CADM1 is a potential tumor suppressor of MPM, consistent with the past characterization of this molecule in other types of tumors. However, in coculture on mesothelial cell monolayers lacking full-length CADM1, CADM1-positive MPM cells spread more widely and grew more quickly, whereas the CADM1-negative cells piled up. Transfection of the CADM1-negative cells with CADM1 cDNA caused them to behave like the CADM1-positive cells, with faster, more widespread growth. These phenotypic differences were not detectable in cocultures on lung fibroblastic monolayers, in which all MPM cells grew much more slowly than on mesothelial cells, irrespective of CADM1 positivity. CADM1 thus appears to mediate efficient adhesion and growth of MPM cells specifically on mesothelial cells, probably via trans-heterophilic binding, and thus may be involved in the manifestation of a considerable subset of MPMs as diffusely growing tumors disseminated over the pleural surface. 相似文献
985.
Haïssaguerre M Derval N Sacher F Jesel L Deisenhofer I de Roy L Pasquié JL Nogami A Babuty D Yli-Mayry S De Chillou C Scanu P Mabo P Matsuo S Probst V Le Scouarnec S Defaye P Schlaepfer J Rostock T Lacroix D Lamaison D Lavergne T Aizawa Y Englund A Anselme F O'Neill M Hocini M Lim KT Knecht S Veenhuyzen GD Bordachar P Chauvin M Jais P Coureau G Chene G Klein GJ Clémenty J 《The New England journal of medicine》2008,358(19):2016-2023
986.
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are Ca2+ channels that localize to intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Recently, IP3Rs were found to participate in the formation of the cytoskeleton and cellular adhesions. In this study, we examined the cellular localization of type I, II, and III IP3Rs to assess their role in cellular adhesion in rat osteoclasts. Rat bone marrow cells were cultured in alpha-MEM with 10% fetal bovine serum, M-CSF, RANKL, and 1,25(OH)2D3 for 1 week to promote osteoclast formation. Type I, II, and III IP3R expression in the osteoclasts was then examined by RT-PCR. Double-staining was performed using antibodies against type I, II, and III IP3Rs and DiOC6, an ER marker, or TRITC-phalloidin, an actin filament marker. Expression of all three IP3Rs was detected in the newly formed osteoclasts; however, the localization of the type I and II IP3Rs was predominantly close to nuclear, and possibly colocalized with the ER, while the type III IP3Rs were localized to the ER and podosomes, actin-rich adhesion structures in osteoclasts. These findings suggest that type III IP3Rs are associated with osteoclast adhesion. 相似文献
987.
Small-angle X-ray diffraction patterns from the anterior byssus retractor muscle of Mytilus edulis in the resting, active, and catch states were examined closely to elucidate the structural features of catch. The specimens were isometrically contracted by stimulation with acetylcholine. The specimens that produced strong tensions in both the active and catch states showed noticeable structural change in the thick filaments. Although the tension was weaker in the catch state than in the active state, the axial spacings of the 14.5 nm meridional reflection and its higher order reflections from the thick filaments were more elongated in the catch state than in the active state. This means that the thick filaments were stretched more strongly in the catch state than in the active state. 相似文献
988.
Shiraishi H Yoshida H Saeki K Miura Y Watanabe S Ishizaki T Hashimoto M Takaesu G Kobayashi T Yoshimura A 《International immunology》2008,20(9):1219-1229
Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules. 相似文献
989.
Kuwahara T Koyama A Koyama S Yoshina S Ren CH Kato T Mitani S Iwatsubo T 《Human molecular genetics》2008,17(19):2997-3009
Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type alpha-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating alpha-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of alpha-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human alpha-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed alpha-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in alpha-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing alpha-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. alpha-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of alpha-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated alpha-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and alpha-synuclein-induced neurotoxicity in synucleinopathy. 相似文献
990.