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1.
Takashi Kojima Norimasa Sawada Yasuo Kokai Masao Yamamoto Michio Mori Yohichi Mochizuki 《Medical Electron Microscopy》1998,31(4):169-176
Occludin, an integral membrane protein, is a candidate for forming the functional intercellular seal of the tight junction. In the present study, we examined the changes of occludin expression and tight junction strand in the cultured hepatocytes. In L-15 medium containing EGF with 2% DMSO and 10–7M glucagon, the great majority of the cells were quiescent. Under this condition, occludin immunoreactivity was observed at cell-to-cell contacts. Tight junction strands formed well-developed networks in freeze-fracture replicas. When the cells entered S phase of the cell cycle, occludin immunoreactivity and the number of tight junction strands decreased. By contrast, when DNA synthesis was then inhibited by readdition of DMSO and glucagon, occludin immunoreactivity and the number of tight junction strands were restored. Regardless of DNA synthesis, however, there was no significant decrease in the protein and mRNA levels of occludin. These changes of occludin immunoreactivity were correlated to the organization of actin filaments. On the other hand, no change of ZO-1 immunoreactivity was observed. These results suggested that tight junctions of cultured hepatocytes regulated in a cell cycle-dependent fashion and correlated with the organization of circumferential actin bundles. 相似文献
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T. Kojima N. Sawada Y. Zhong M. Oyamada M. Mori 《Virchows Archiv : an international journal of pathology》1994,425(4):407-412
Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions. 相似文献
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目的探讨非小细胞肺癌中磷脂酰肌醇-3-激酶(PI3K)/AKT和丝裂原活化蛋白激酶MAPK/ERK信号通路对Forkhead转录因子(Fox蛋白家族)FOXO1活性的影响及分子机制。方法体外培养人非小细胞肺癌系A549,分别选用PI3K/AKT信号通路特异性抑制剂LY294002和MAPK/ERK信号通路特异性抑制剂UO126及两种抑制剂联合处理细胞之后,MTT比色法检测其对细胞增殖的影响;Western blot检测蛋白FOXO1、p-FOXO1及信号下游蛋白Bim表达的变化;免疫荧光法检测FOXO1蛋白在A549细胞中亚细胞的定位的变化。结果与对照组相比,LY294002和UO126都能明显地抑制A549细胞的增殖,且具有时间依赖性。Western blot结果显示,FOXO1的蛋白水平未见显著性变化,而FOXO1的磷酸化水平明显下降,Bim的表达相应增加。免疫荧光结果显示,FOXO1在A549细胞中的核转位增多。LY294002和UO126联合处理A549细胞时,较药物单独作用效果明显增加。结论 PI3K/AKT和MAPK/ERK信号通路能调节FOXO1磷酸化水平,且具有协同作用,抑制其转录活性。FOXO1通过激活Bim蛋白,促进细胞凋亡,抑制细胞的增殖。 相似文献
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Caspases are instrumental in the implementation of apoptotic cell death, and caspase activation is in most investigated cases closely linked to apoptosis. Recent data demonstrate, however, that caspases are also activated during primary T cell activation in the absence of apoptosis. Here we provide evidence that caspase activity is required for some but not all aspects of T cell activation. CD3-triggered proliferation of mouse T cells was impaired in the presence of the pan-caspase-inhibitor Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) and the number of cells entering the cell cycle was reduced. Costimulation by CD28 or externally added interleukin-2 (IL-2) failed to rescue proliferation. Re-stimulation of pre-activated T cells, however, was not affected by Z-VAD-fmk. Intriguingly, CD3-induced production of IL-2 by primary T cells was not impaired in the presence of Z-VAD-fmk. Likewise, CD3-induced activation of mitogen-activated protein kinases was unaffected by Z-VAD-fmk and intracellular levels of inhibitory kappaBalpha were the same as in control cells. T cells transgenically expressing a dominant negative mutant of the caspase-adaptor Fas-associated molecule with death domain (FADD)/MORT1 displayed the same pattern of reaction, i.e. a reduced proliferative response but normal IL-2-production. These data show a distinct role of caspases during primaryT cell activation and provide evidence for a FADD-caspase-pathway not only in the induction of apoptosis but also of T cell proliferation. 相似文献
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目的本实验利用APP/PS1双转基因阿尔茨海默病(AIzheimer disease,AD)小鼠模型,观察盐酸多奈派齐对AD小鼠学习记忆能力及海马磷脂酰肌醇3激酶(phosphoinositide 3-kinases,PI3K)表达的影响。方法 APP/PS1双转基因模型小鼠20只,随机分为AD模型组(10)和盐酸多奈派齐组(10),再取同窝阴性小鼠10只,作为对照组。经跳台试验和水迷宫试验进行行为学测试,用免疫组化方法检测各组小鼠海马PI3K的表达变化。结果与对照组相比,AD模型组小鼠的学习和记忆成绩明显降低(<0.05);相比于AD模型组小鼠,盐酸多奈哌齐组小鼠的学习和记忆成绩明显提高(<0.05)。免疫组化检测结果证明,盐酸多奈哌齐组小鼠和对照组小鼠海马PI3K蛋白阳性表达明显增加,平均光密度值分别为(0.48±0.19)和(0.74±0.22),显著高于AD模型组(0.31±0.10,<0.05)。Western blot结果发现,盐酸多奈哌齐组小鼠和对照组小鼠海马PI3K蛋白的平均光密度值分别为(0.39±0.09)和(0.61±0.21),显著高于AD模型组(0.26±0.07,<0.05)。结论 AD小鼠海马区PI3K的表达上调可能是盐酸多奈哌齐改善AD小鼠学习和记忆功能的机制之一。 相似文献
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David Jackson Marian J. Killip Caroline S. Galloway Rupert J. Russell Richard E. Randall 《Virology》2010,396(1):94-105
A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K. 相似文献
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Lugli A Zlobec I Minoo P Baker K Tornillo L Terracciano L Jass JR 《Human pathology》2006,37(8):1022-1031
Tumor budding is defined as dedifferentiated cancer cells at the invasive margin of colorectal cancer (CRC) and correlates with a worse prognosis. The invasive margin and tumor budding are normally not present in a superficial diagnostic biopsy specimen. The aim of this study was to investigate the expression/overexpression of 2 downstream molecules of the mitogen-activated protein kinase and phosphoinositide 3-kinase/AKT pathways, phosphorylated AKT (pAKT) and phosphorylated extracellular signal-regulated kinase (pERK), in areas of CRC away from the invasive margin and determining if these variables were predictive of tumor budding or prognosis. A series of 1420 unselected, nonconsecutive CRC resections were subdivided into 3 groups: (1) DNA mismatch repair (MMR) proficient, (2) MLH1-negative, and (3) presumed HNPCC. Immunohistochemical analysis of pAKT and pERK expression (0% versus > 0%) and overexpression (increasing percentage of positivity) was performed using the tissue microarray technique. The results were correlated with clinicopathologic parameters. Fifty-seven samples of normal colon mucosa were included as a control group. Nuclear pERK expression (P = .008) was associated with presence of tumor budding in the MMR-proficient, but not in the MLH1-negative and presumed-HNPCC groups. In contrast, cytoplasmic pAKT overexpression was associated with early T stage (P = .04), early N stage (P = .02), and absence of tumor budding (P = .03) only in the MLH1-negative group. There was no association between pERK or pAKT and clinicopathologic parameters in the HNPCC group. Dysregulation of the mitogen-activated protein kinase pathway is likely to be implicated in the mechanism of tumor budding only in MMR-proficient CRC, whereas the phosphoinositide 3-kinase/AKT pathway is associated with early stage in MLH1-negative CRC. 相似文献
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Haiming Wang Xingwang Yang 《International journal of clinical and experimental pathology》2015,8(1):881-887
Recently, there is growing evidence that tight junction proteins are often abnormally regulated in human tumors. The function of tight junction proteins in the maintenance of normal epithelial physiology has been well discussed, but their role in the tumorigenesis of gastric cancer is less well defined. To explore the expression distinction of the tight junction proteins claudin-1, -3, and -4 expression in the gastric cancer, the expression of claudin-1, -3, and -4 in 92 gastric cancer tissues and the non-neoplastic tissues adjacent to the tumors were examined by immunohistochemistry. Compared with adjacent non-neoplastic tissues, the expression of claudin-1 was down regulated. However, the expression of claudin-3 and claudin-4 were up-regulated in gastric cancer tissue. In addition, the expression of claudin-3 is correlated with claudin-4 expression in gastric cancer. Our present study reveals that claudin-1, -3, and -4 protein expression altered between human gastric cancers and adjacent non-neoplastic tissues. 相似文献
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目的 研究乙醇对原代肝细胞中P70S6K和ERK1/2活性的影响。方法 分离成年雄性SD大鼠的肝细胞,分别以0、50、100 和200mmol/L的乙醇处理4h,或以200 mmol/L乙醇处理0、1、2和4h,用Western blot法检测P70S6K和ERK1/2的活性。在细胞中加入100 nmol/L的mTOR特异抑制剂雷帕霉素或10μmol/L 的MEK1/2特异抑制剂U0126,培养 24 h后收集细胞,用Western blot法检测P70S6K、ERK1/2及PPARγ的表达;用EMSA检测PPARγ的活性。结果 随着乙醇浓度的增加或处理时间的延长,P70S6K的表达逐渐降低;乙醇也可抑制ERK1/2的活性;U0126同时抑制ERK1/2和P70S6K的活性,而雷帕霉素虽抑制P70S6K的活性,但增强ERK1/2的活性;乙醇不影响PPARγ的表达和活性。 结论 在大鼠原代肝细胞,乙醇对P70S6K的活性有抑制作用,且存在量效关系和时效关系。P70S6K的活性受ERK1/2的控制,但P70S6K也可反向调节ERK1/2。 相似文献
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《Expert Review of Clinical Immunology》2013,9(8):1107-1123
Metastatic malignant melanoma is a frequently fatal cancer. In recent years substantial therapeutic progress has occurred with the development of targeted kinase inhibitors and immunotherapeutics. Targeted therapies often result in rapid clinical benefit however responses are seldom durable. Immune therapies can result in durable disease control but responses may not be immediate. Optimal cancer therapy requires both rapid and durable cancer control and this can likely best be achieved by combining targeted therapies with immunotherapeutics. To achieve this, a detailed understanding of the immune consequences of the various kinase inhibitors, in development, clinical trial and currently used to treat melanoma is required. 相似文献
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Lina Qi Jingle Jiang Pengjin Jin Meiqian Kuang Quanwei Wei Fangxiong Shi Dagan Mao 《Acta histochemica》2018,120(3):221-227
To study the expression patterns of claudin-5 and its related signals during luteal regression in rats, a sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpus luteum (CL). A total of 35 rats were treated with one PGF or two PGF at an interval of 24?h from day 7 of pseudopregnancy to induce CL regression. Serum and ovaries were collected at 0, 2, 4, 8 or 24?h after one PGF injection (1 PGF), 2 or 24?h after two PGF injections (2 PGF). The serum progesterone level was detected by RIA; the ovarian expression of claudin-5, the phosphorylations of STAT3 (p-STAT3), Akt (p-Akt), ERK1/2 (p-ERK) and p38 MAPK (p-p38) were detected by western blot, real-time PCR and IHC. Results showed that serum progesterone (P4) decreased after PGF treatment. Claudin-5 mRNA decreased at 4?h and 8?h after 1 PGF and 2?h after 2 PGF, and claudin-5 protein decreased at 4?h after 1 PGF. p-STAT3 increased at 4?h after 1 PGF and 2?h after 2 PGF. p-ERK increased at 2?h after 2 PGF. The level of p-Akt decreased at 4?h after 1 PGF. PGF treatment did not alter the phosphorylation of p38 MAPK at any time points in this study. IHC results revealed that claudin-5 was expressed in the nuclei and cytoplasm of steroidogenic cells and in the vessels, while PGF induced-p-STAT3 was expressed uniformly in the cytoplasm of luteal steroidogenic cells. In conclusion, PGF treatment decreased the expression of claudin-5 and the additional PGF treatment enhanced the decrease in claudin-5 mRNA expression and the increases in ERK1/2 and STAT3 phosphorylation in the corpus luteum of pseudopregnant rats, which will contribute new information to the further study of molecular mechanism of luteal regression. 相似文献
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Rucinski M Ziolkowska A Hochol A Pucher A Macchi C Belloni AS Nussdorfer GG Malendowicz LK 《International journal of molecular medicine》2006,18(4):565-570
Evidence is available that some endocrine disruptors, acting as selective estrogen receptor modulators (SERMs), interfere with osteoblast differentiation and function. Therefore, we investigated whether 17beta-estradiol, bisphenol-A (BSP), silymarin, genistein, resveratrol, procymidone, linurone and benzophenone-3 (BP3) modulate differentiation of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. Disruptors were added at day 18 of culture and cells were harvested 48 h later. Real time-PCR revealed that estradiol and resveratrol enhanced osteocalcin mRNA expression in ROB cells, while other disruptors were ineffective. The expression of osteonectin and collagen-1alpha was not affected by any disruptor. Estradiol, resveratrol, genistein and BSP stimulated the proliferative activity of ROB cells. In contrast, procymidone and linurone inhibited the proliferative activity, and silymarin and BP3 were ineffective. The conclusion is drawn that i) only resveratrol is able, like estradiol, to stimulate the specialized functions of ROB cells, and ii) the proliferative activity of ROB cells is more sensitive to endocrine disruptors, some of which could probably act via a mechanism independent of their SERM activity. 相似文献
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《Immunopharmacology and immunotoxicology》2013,35(5):810-814
Antimycin A (AMA) inhibits mitochondrial electron transport chain between cytochrome b and c. In the present study, we investigated the effects of dehydrocostus lactone on osteoblastic MC3T3-E1 cells in the presence of AMA with a focus on redox changes and PI3K/Akt/CREB signaling. AMA increased nitrotyrosin level and decreased NADPH level, activities of thioredoxin reductase, phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B [PKB]), and phosphorylated cAMP-response element-binding protein (CREB). Pretreatment with dehydrocostus lactone prior to AMA exposure significantly prevented the loss of NADPH, production of nitrotyrosine, and thioredoxin reductase inactivation induced by AMA. Moreover, dehydrocostus lactone increased activities of PI3K and Akt, and CREB phosphorylation inhibited by AMA. These results suggest that antioxidant activity and PI3K/Akt/CREB activation are related to the protective effect of dehydrocostus lactone against osteoblast damage induced by AMA. 相似文献
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Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H2O2 as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H2O2 treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H2O2 exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H2O2-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons. 相似文献
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Qiu Qin Ping Liu Lin Liu Rong Wang Ni Yan Jing Yang Xuan Wang Madhu Pandey Jin-an Zhang 《Brazilian journal of medical and biological research》2012,45(12):1202-1208
Hashimoto''s thyroiditis (HT) is considered to be mediated mainly by Th1 cells, but it is not known whether Graves'' disease (GD) is associated with Th1 or Th2 predominance. Th17 cells, a novel subset of Th cells, play a crucial role in the pathogenesis of various autoimmune disorders. In the present study, the expression of IL-17A and IFN-γ was investigated in patients with HT or GD. mRNA expression of IL-17A and IFN-γ in peripheral blood mononuclear cells (PBMC) from 43 patients with autoimmune thyroid disease (AITD) and in thyroid tissues from 40 AITD patients were measured by real-time quantitative PCR. The protein expression of IL-17A and IL-23p19 was examined by immunohistochemistry in thyroid tissues from 28 AITD patients. The mRNA levels of IL-17A and IFN-γ were higher in both PBMC and thyroid tissues of HT patients than in controls (mRNA levels are reported as the cytokine/β-actin ratio: IL-17 = 13.58- and 2.88-fold change and IFN-γ = 16.54- and 2.74-fold change, respectively, P < 0.05). Also, the mRNA levels of IL-17A and IFN-γ did not differ significantly in GD patients (P > 0.05). The high protein expression of IL-17A (IOD = 15.17 ± 4.8) and IL-23p19 (IOD = 16.84 ± 7.87) in HT was confirmed by immunohistochemistry (P < 0.05). The similar high levels of IL-17A and IFN-γ suggest a mixed response of Th17 and Th1 in HT, where both cells may play important roles in the destruction procedure by cell-mediated cytotoxicity. 相似文献