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61.
Maruyama H Higuchi N Nishikawa Y Kameda S Iino N Kazama JJ Takahashi N Sugawa M Hanawa H Tada N Miyazaki J Gejyo F 《The journal of gene medicine》2002,4(3):333-341
Background
High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.Methods
We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.Results
We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.Conclusions
These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.62.
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The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells. 相似文献
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Tokuo Saito Takamasa Ueno Tomoko Kubori Shigeru Yamaguchi Tetsuo Iino & Shin-Ichi Aizawa 《Molecular microbiology》1998,27(6):1129-1139
Among motile revertants isolated from flagellar hook-deficient ( flgE ) mutants of Salmonella typhimurium one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro . This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln→Ala-Arg) in the C-terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook–basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments. 相似文献
68.
Manami Miyai Shingo Eikawa Akihiro Hosoi Tamaki Iino Hirokazu Matsushita Midori Isobe Akiko Uenaka Heiichiro Udono Jun Nakajima Eiichi Nakayama Kazuhiro Kakimi 《PloS one》2015,10(8)
Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring. 相似文献
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F1-ATPase is a molecular motor in which the γ subunit rotates inside the α3β3 ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F1-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly
depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F1 and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important
issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe
could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study,
we assessed the stiffness of two different portions of F1 from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α3β3 ring, and the complex of the external part of the γ subunit and the α3β3 ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the
rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian,
respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit
is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also
partially explains the accommodation of the intrinsic step size mismatch between Fo and F1-ATPase. 相似文献