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31.
We investigated the neuroprotective function of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) after transient hypoxia (12 h) and reoxygenation (0-72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG-tagged alpha7nAChR (alpha7pCMV cells) and control PC12 cells (non-transfected or transfected with vector only) in medium with and without nicotine. Plasma membrane degradation in the early phase after hypoxia was inhibited in PC12 cells with nicotine, and more profoundly in alpha7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in alpha7pCMV cells with nicotine, but, surprisingly, it was more remarkable in alpha7pCMV cells without nicotine than in PC12 cells with nicotine. G1-arrest of the cell cycle, observed in control PC12 cells at 12 h after hypoxia, preceding DNA fragmentation, was not evident in alpha7pCMV cells, with or without nicotine. Furthermore, in alpha7pCMV cells with and without nicotine, the basal expression levels of total Akt were approximately 1.5-fold higher, and the up-regulation of Akt phosphorylated at Ser473 after hypoxia was strikingly enhanced, compared with control PC12 cells. These findings suggest that alpha7nAChR functions constitutively in PC12 cells, that its overexpression raises tolerance against G1-arrest and DNA fragmentation after hypoxia, and that it can be considered a candidate target for treatment against hypoxia-induced acute membrane degradation and delayed DNA fragmentation in neurons. 相似文献
32.
Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability 总被引:1,自引:0,他引:1
Funahashi J Takano K Yamagata Y Yutani K 《The Journal of biological chemistry》2002,277(24):21792-21800
Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein. 相似文献
33.
Impaired proliferative response of V alpha 24 NKT cells from cancer patients against alpha-galactosylceramide 总被引:5,自引:0,他引:5
Yanagisawa K Seino K Ishikawa Y Nozue M Todoroki T Fukao K 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(12):6494-6499
Human invariant V alpha 24(+) NKT cells are a relatively new subpopulation of lymphocytes. It has been reported that V alpha 24 NKT cells are significantly involved in some human diseases. We have evaluated the number and function of V alpha 24 NKT cells in both healthy volunteers and cancer patients. In this study we found that V alpha 24 NKT cells in unfractionated PBMCs obtained from cancer patients did not respond efficiently to alpha-galactosylceramide (alpha-GalCer) in vitro. Thus, their proportion after stimulation with alpha-GalCer was smaller than that found in healthy volunteers. However, the cancer patients' V alpha 24 NKT cells retained cytotoxic activity against malignant target cells, and they could efficiently proliferate to alpha-GalCer when fractionated by sorting. Furthermore, we found that addition of G-CSF to the culture could restore the low proliferative response of V alpha 24 NKT cells from cancer patients. These results suggest that some functions of NKT cells in cancer patients are impaired, and this observation carries significant implications for immunotherapy-based cancer treatments. 相似文献
34.
Ito Y Arai S van Oers NS Aifantis I von Boehmer H Miyazaki T 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(9):4913-4919
It has been of much interest whether there is functional redundancy between the constitutively signaling pre-Talpha/TCRbeta (pre-TCR) and ligated TCRalphabeta complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in beta-selection at the CD4(-)CD8(-) double-negative stage and the TCRalphabeta being crucial for positive/negative selection at the CD4(+)CD8(+) double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCRalpha-deficient (TCRalpha(-/-)) mice produced a small number of mature CD8(+) T cells. Surprisingly, when pre-Talpha was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8(+) T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8(+) T cells was also observed in TCRalpha(-/-) mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8(+) T cells drastically decreased in the absence of pre-Talpha on a TCRalpha(-/-) background. Thus, the pre-TCR appears to functionally promote positive selection of CD8(+) T cells. The biased production of CD8(+) T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment. 相似文献
35.
Huang J Wu L Yalda D Adkins Y Kelleher SL Crane M Lonnerdal B Rodriguez RL Huang N 《Transgenic research》2002,11(3):229-239
Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed. 相似文献
36.
Rong S Ikeda H Sato Y Hirohashi Y Takamura Y Sahara H Sato T Maeda A Ohguro H Sato N 《Cancer immunology, immunotherapy : CII》2002,51(5):282-290
Recoverin is a photoreceptor-specific calcium binding protein that is only expressed in the retina in normal tissues. Aberrant expression of recoverin, however, has been observed in several cancer tissues and may cause a very rare autoimmune disease, cancer-associated retinopathy (CAR). It has been suggested that CAR-positive cancer patients have a more favorable prognosis than CAR-negative cancer patients. To estimate the status of recoverin-specific T cells in such cancer patients, we generated an HLA-A24-recoverin peptide tetramer. By use of the tetramer, we could directly assess the frequency of CTL precursors (CTLp) of 20 HLA-A24(+) cancer patients with ten colon, six stomach and four breast cancers, and seven healthy individuals. Four cancer patients showed a CTLp frequency higher than 0.9%. Seven cancer patients including the former four patients and two healthy individuals showed specific anti-recoverin cytotoxic responses in an HLA-A24-restricted manner after in vitro stimulation with the recoverin peptide. Moreover, five cancer patients analyzed in an independent experiment using different peripheral blood mononuclear cells (PBMC) samples showed similar CTLp and cytotoxic T lymphocyte (CTL) frequencies and cytotoxic responses, suggesting that the CTLp frequency analyzed by the tetramer and the cytotoxic response may have a good correlation. Thus, we hypothesize that anti-recoverin CTLp may exist in some cancer patients, and that anti-recoverin CTL may be readily induced. 相似文献
37.
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39.
Matsumura K Abe I Tsuchihashi T Fujishima M 《American journal of physiology. Regulatory, integrative and comparative physiology》2000,278(5):R1314-R1320
We determined the cardiovascular and neurohormonal responses to intracerebroventricular injection of leptin in conscious rabbits. Intracerebroventricular injection of leptin elicited dose-related increases in mean arterial pressure and renal sympathetic nerve activity while producing no consistent, significant increases in heart rate. Peak values of mean arterial pressure and renal sympathetic nerve activity induced by intracerebroventricular injection of 50 microgram of leptin (+17.3 +/- 1.2 mmHg and +47.9 +/- 12.0%) were obtained at 10 and 20 min after injection, respectively. Plasma catecholamine concentrations significantly increased at 60 min after intracerebroventricular injection of leptin (control vs. 60 min; epinephrine: 33 +/- 12 vs. 97 +/- 27 pg/ml, P < 0.05; norepinephrine: 298 +/- 39 vs. 503 +/- 86 pg/ml, P < 0.05). Intracerebroventricular injection of leptin also caused significant increases in plasma vasopressin and glucose levels. However, pretreatment with intravenous injection of pentolinium (5 mg/kg), a ganglion blocking agent, abolished these cardiovascular and neurohormonal responses. On the other hand, intravenous injection of the same dose of leptin (50 microgram) as used in the intracerebroventricular experiment failed to cause any cardiovascular and renal sympathetic nerve responses. These results suggest that intracerebroventricular leptin acts in the central nervous system and activates sympathoadrenal outflow, resulting in increases in arterial pressure and plasma glucose levels in conscious rabbits. 相似文献
40.
Yuriko Uehara Katsutoshi Oda Yuji Ikeda Takahiro Koso Shingo Tsuji Shogo Yamamoto Kayo Asada Kenbun Sone Reiko Kurikawa Chinami Makii Otoe Hagiwara Michihiro Tanikawa Daichi Maeda Kosei Hasegawa Shunsuke Nakagawa Osamu Wada-Hiraike Kei Kawana Masashi Fukayama Keiichi Fujiwara Tetsu Yano Yutaka Osuga Tomoyuki Fujii Hiroyuki Aburatani 《PloS one》2015,10(6)
Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. 相似文献