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1

Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA  

PubMed Central

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

2013-01-01

2

Human immunodeficiency virus type 1 envelope glycoprotein gp120 produces immune defects in CD4+ T lymphocytes by inhibiting interleukin 2 mRNA.  

PubMed Central

Envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) is known to inhibit T-cell function, but little is known about the mechanisms of this immunosuppression. Pretreatment of a CD4+ tetanus toxoid-specific T-cell clone with soluble gp120 was found to exert a dose-dependent inhibition of soluble antigen-driven or anti-CD3 monoclonal antibody-driven proliferative response, interleukin 2 (IL-2) production, and surface IL-2 receptor (IL-2R) alpha-chain expression, all of which were reversed by the addition of exogenous IL-2. mRNA for the gene encoding IL-2 was suppressed by treatment with gp120, but IL-2R gene transcription was not inhibited. Bypass activation of the T-cell clone with phorbol 12-myristate 13-acetate plus ionomycin was unaffected by gp120 pretreatment. Thus, gp120-CD4 interaction interferes with an essential role of the CD4 molecule in signal transduction through the CD3-antigen receptor (Ti) complex. Such a mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed specific immune responses associated with HIV infection. Images PMID:2315327

Oyaizu, N; Chirmule, N; Kalyanaraman, V S; Hall, W W; Pahwa, R; Shuster, M; Pahwa, S

1990-01-01

3

Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA.  

PubMed

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

2013-01-01

4

BNIP3 upregulation by ERK and JNK mediates cadmium-induced necrosis in neuronal cells.  

PubMed

Cadmium (Cd) is a toxic heavy metal that may cause neurological disorders. We studied the mechanism underlying Cd-mediated cell death in neuronal cells. In Cd-induced neurotoxicity, caspase-3 was only modestly activated, and accordingly, zVAD-fmk, a pan-caspase inhibitor, partially attenuated cell death. However, pretreatment with Necrox-2 or Necrox-5, two novel necrosis inhibitors, suppressed cell death more markedly compared with pretreatment with zVAD-fmk. Moreover, the necrosis inhibitors did not prevent cleavage of caspase-3. These results indicate that caspase-independent necrosis is more prevalent in Cd-induced neurotoxicity. Bcl-2 and adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) has been reported to be related to caspase-independent cell death. Cd treatment caused a dramatic upregulation of BNIP3 mRNA and protein levels in vitro and in vivo. Furthermore, knockdown of BNIP3 greatly inhibited Cd-induced cell death. Importantly, BNIP3 RNAi decreased lactate dehydrogenase release and the percentage of propidium iodide-positive cells, two markers of necrotic cell death due to rupture of the cell membrane, whereas it had no effect on activation of caspase-3 in Cd-treated cells. These data suggest that BNIP3 mediates caspase-independent necrosis, but not apoptosis. Moreover, our results indicate that induction of BNIP3 by Cd may not be related to HIF-1 which is generally regarded as a mediator responsible for BNIP3 expression. Finally, we show that mitogen-activated protein kinases (MAPKs) are activated by Cd in vitro and in vivo; ERK and JNK promote BNIP3 upregulation and subsequent necrosis. Taken together, our results suggest BNIP3, upregulated by activation of ERK and JNK, mediates Cd-induced necrosis in neuronal cells. PMID:24824807

Wang, Bin; Xiao, Jia-Li; Ling, Yi-Hui; Meng, Xiao-Jing; Wu, Bing; Yang, Xin-Yi; Zou, Fei

2014-08-01

5

Effects of selenium on proliferation, interleukin-2 production and selenoprotein mRNA expression of normal and dexamethasone-treated porcine splenocytes.  

PubMed

Porcine splenocytes were isolated in vitro, treated with different levels of dexamethasone (DEX), and stimulated by concanavalin A. Further, the normal (non-DEX-supplemented) or DEX-treated (0.01?µmol/L) splenocytes were incubated with 0, 0.5, 2, and 5?µmol/L Na2SeO3. The splenocyte proliferation, IL-2 production, intracellular glutathione peroxidase 1 (GPx1) mRNA level and activity and thioredoxin reductase 1 mRNA level were measured. The results showed that addition of 0.5 or 2?µmol/L Na2SeO3 significantly promoted normal and DEX-treated splenocyte proliferation, IL-2 production and GPx1 mRNA expression and activity (P?

Zhuang, Tenghan; Xu, Haibin; Hao, Shu; Ren, Fei; Chen, Xingxiang; Pan, Cuiling; Huang, Kehe

2015-02-01

6

Structure of the Human Interleukin2 Receptor Gene  

Microsoft Academic Search

The gene encoding the human interleukin-2 (IL-2) receptor consists of 8 exons spanning more than 25 kilobases on chromosome 10. Exons 2 and 4 were derived from a gene duplication event and unexpectedly also are homologous to the recognition domain of human complement factor B. Alternative messenger RNA (mRNA) splicing may delete exon 4 sequences, resulting in a mRNA that

Warren J. Leonard; Joel M. Depper; Minoru Kanehisa; Martin Kronke; Nancy J. Peffer; Penny B. Svetlik; Margery Sullivan; Warner C. Greene

1985-01-01

7

DNA damage during mitosis invokes a JNK-mediated stress response that leads to cell death.  

PubMed

Mitotic catastrophe is a phenomenon displayed by cells undergoing aberrant mitosis to eliminate cells that fail to repair the errors. Why and how mitotic catastrophe would lead to cell death remains to be resolved and the answer will prove valuable in design of better therapeutic agents that specifically target such cells in mitosis. The antibiotic actinomycin D has been shown to induce chromosomal lesions in lower order organisms as well as in human interphase cells. Relatively few studies have been conducted to elucidate molecular events in the context of mitotic DNA damage. We have previously established a model of mitotic catastrophe in human HeLa cells induced by actinomycin D. Here, we show that actinomycin D induce cellular stress via DNA damage during mitosis. The higher order packing of chromosomes during mitosis might impede efficient DNA repair. gammaH2AX serves as a marker for DNA repair and active JNK interacts with gammaH2AX in actinomycin D-treated mitotic extracts. We believe JNK might be in part, responsible for the phosphorylation of H2AX and thereby, facilitate the propagation of a positive signal for cell death, when repair is not achieved. The mitotic cell activates JNK-mediated cell death response that progresses through a caspase cascade downstream of the mitochondria. In the mean time, remaining checkpoint signals may be sufficient to put a restraining hand on entry into anaphase and the cell eventually dies in mitosis. PMID:20512932

Ho, Chin-Yee; Li, Hoi-Yeung

2010-06-01

8

Molecular cloning and expression of cDNAs for the human interleukin-2 receptor  

Microsoft Academic Search

We have purified the human T-cell growth factor (interleukin-2) receptor and have cloned, sequenced and expressed cDNAs corresponding to this receptor. We identify one gene, but two interleukin-2 receptor mRNAs which differ in their polyadenylation signals. We have isolated an additional cDNA that may correspond to an alternatively spliced mRNA that lacks a 216 base segment and appears to encode

Warren J. Leonard; Joel M. Depper; Gerald R. Crabtree; Stuart Rudikoff; Janet Pumphrey; Richard J. Robb; Martin Krönke; Penny B. Svetlik; Nancy J. Peffer; Thomas A. Waldmann; Warner C. Greene

1984-01-01

9

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways  

PubMed Central

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G2/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45?. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-? or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. PMID:22285274

Ngoc, Tam Dan Nguyen; Son, Young-Ok; Lim, Shin-Saeng; Shi, Xianglin; Kim, Jong-Ghee; Heo, Jung Sun; Choe, Youngji; Jeon, Young-Mi; Lee, Jeong-Chae

2012-01-01

10

Bornyl caffeate induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways  

PubMed Central

Aim: To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms. Methods: Human breast cancer cell line MCF-7 and other tumor cell lines (T47D, HepG2, HeLa, and PC12) were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential (MMP) was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species (ROS) were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis. Results: Bornyl caffeate (10, 25, and 50 ?mol/L) suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 ?mol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), z-VAD (pan-caspase inhibitor) or the thiol antioxidant L-NAC. Conclusion: Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways. PMID:24335836

Yang, Chuan-bin; Pei, Wei-jing; Zhao, Jia; Cheng, Yuan-yuan; Zheng, Xiao-hui; Rong, Jian-hui

2014-01-01

11

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways  

SciTech Connect

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45?. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-? or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ? The mode of NaF-induced cell death and the mechanisms involved were examined. ? NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ? NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ? JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ? ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

Nguyen Ngoc, Tam Dan [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of)] [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Son, Young-Ok [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States)] [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lim, Shin-Saeng [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of) [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States)] [Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Kim, Jong-Ghee [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of)] [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Heo, Jung Sun [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of)] [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Choe, Youngji [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of)] [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Jeon, Young-Mi, E-mail: young@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of)] [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of) [Institute of Oral Biosciences and School of Dentistry (BK21 Program), Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Graduate Center for Toxicology, School of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

2012-03-15

12

Human pregnancy serum inhibits interleukin-2 production.  

PubMed Central

Cell-mediated immunity may be depressed during pregnancy. We used the two way mixed lymphocyte reaction as an in vitro model of cell mediated immunity and studied the effect of pregnancy sera on this system by the amount of tritiated thymidine taken up by activated lymphocytes. We found that: (1) pregnancy sera contain a factor inhibiting the mixed lymphocyte reaction; (2) the inhibition of the mixed lymphocyte reaction induced by sera could be reversed by the addition of the supernatant from allogeneic mixed lymphocyte reaction; (3) pure interleukin-1 could not reverse the inhibitory effect and (4) recombinant interleukin-2 (IL-2) completely reversed the inhibitory effect of pregnancy sera on the mixed lymphocyte reaction. We conclude that a factor (or factors) present in serum from pregnant women is capable of inhibiting the generation of IL-2 during lymphocyte activation. PMID:6239719

Nicholas, N S; Panayi, G S; Nouri, A M

1984-01-01

13

Interleukin 2, soluble interleukin 2 receptor, and interferon-? in the suction blister fluids from psoriatic skin  

Microsoft Academic Search

Psoriasis represents inflammatory skin disorders characterized by significant changes in cellular immunity, particularly exhibiting alterations in T lymphocyte-related functions. Early psoriatic lesions have been reported to show an infiltration of activated helper T cells. Elevated levels of interleukin 2 (IL-2), IL-2 receptor (IL-2R), and interferon-? (INF-?) are associated with an early activation of T cells. To examine local activation of

H. Takematsu; H. Tagami

1990-01-01

14

Interleukin-2 receptor expression in human mast cells and basophils.  

PubMed

Surgical human thymus, upper respiratory tract, lung and small and large bowel specimens were analyzed for the presence of interleukin 2 receptor (IL2-R)-positive cells. Histochemical (toluidine) and immunologic (anti-IL2-R monoclonal antibody) staining procedures revealed a distinct anti-IL2-R positivity in most of metachromatically staining cells. These positive cells were observed not only in tissues showing strong inflammatory reaction and mast cell hyperplasia, as in Crohn's disease, but also in those not histologically affected by pathologic conditions. This finding suggested that human mast cells, like T blast cells, express the p55 chain of IL2-R on their surface. To see whether IL2-R was being actively synthesized, a cell preparation rich in peripheral blood basophils (PBB), which are cells closely related to mast cells, was obtained. Ultrastructural analysis of PBB after indirect immunogold procedure revealed that the vast majority expressed the IL2-R. Moreover, the presence of intracellular reaction products in the cytoplasm of most membrane-positive PBB was indicative of active antigen synthesis. Furthermore, Northern blot analysis evidenced specific IL2-R mRNA in PBB, while its expression was augmented several times when PBB were cultured in the presence of stimulated T cell supernatant. PMID:2312185

Maggiano, N; Colotta, F; Castellino, F; Ricci, R; Valitutti, S; Larocca, L M; Musiani, P

1990-01-01

15

Characterization of dog interleukin-2 activity.  

PubMed

Proliferative activity of murine interleukin-2 (IL-2) dependent T cells (CTLL-2) was detected in the culture supernatant of canine peripheral blood lymphocytes (PBL) stimulated with phytohemagglutinin-P (PHA-P), and was defined as dog IL-2. The highest production of IL-2 was obtained under the conditions in which a PBL population of 2 x 10(6) cells/ml was stimulated with PHA-P at a concentration of 10 micrograms/ml at 38 degrees C for 48 hr. Dog IL-2 activity was significantly inhibited by heating at 65 degrees C, acidification under pH 4, alkalification over pH 10, and trypsin exposure. A peak of dog IL-2 activity was detected in the fraction with a molecular weight of approximately 31,000 by gel filtration. Long-term culture of canine lymphocytes was successful over 10 passages by using dog IL-2 with PHA-P-stimulation every 3 passages. The cultured cells mostly consisted of small- and medium-sized lymphocytes. These cells reacted to anti-dog thymocyte rabbit serum and anti-dog Thy-1 mouse monoclonal antibody. These cells were therefore considered to originate in T-lineage lymphocytes. Cytostasis of PBL from intact dogs reacting to canine transmissible venereal sarcoma cells was increased significantly when PBL was cultured for more than 30 days with homologous IL-2. PMID:8117817

Mizuno, S; Fujinaga, T; Hagio, M

1993-12-01

16

Original article Human recombinant interleukin-2 augments porcine  

E-print Network

Original article Human recombinant interleukin-2 augments porcine natural killer cell cytotoxicity 1989) Summary ― Immunological parameters of porcine peripheral blood mononuclear cells after bacterin on day 0. Cytolytic activity to porcine fibroblasts (PK-15) was increased (P

Boyer, Edmond

17

Nicotine exaggerates LPS-induced airway hyperreactivity via JNK-mediated up-regulation of Toll-like receptor 4.  

PubMed

Tobacco smokers often display increased airway hyperreactivity (AHR) when faced with bacterial infections. The present study uses a murine organ-culture model to dissect the mechanisms involved in this exaggerated smooth muscle response. Nicotine simulates the effects of smoking, and LPS represents bacterial infection. Contractile responses of isolated murine tracheal segments were analyzed in myographs after organ culture with increasing concentrations of LPS and/or nicotine for 4 days with or without specific MAPK inhibitors. Nicotine's effect on the expression of cell surface Toll-like receptors (TLRs), MCP-1, COX-2, and TNF-? were examined by real-time PCR. Increased protein expression was verified by immunohistochemistry. LPS concentration-dependently increased contractile responses to bradykinin and des-Arg(9)-bradykinin. A combination of nicotine and low-dose LPS caused powerful synergistic contractions along with increased kinin receptor expression. Specific kinin B1 and B2 receptor inhibitors blocked this reaction. Nicotine increased mRNA and protein expression of TLR4 and -6 in the epithelium and smooth muscle layer, with MCP-1 and COX-2 mRNA increasing in parallel. Specific inhibition of JNK attenuated nicotine's effects. In conclusion, long-term exposure to nicotine up-regulated the expression of TLR4 and -6 via a JNK-related pathway, causing an exaggeration of the LPS-induced local airway inflammation and increased AHR. This might offer a mechanistic explanation to the increased AHR seen in tobacco smokers confronted with bacterial infections. PMID:24669857

Xu, Yuan; Zhang, Yaping; Cardell, Lars-Olaf

2014-09-01

18

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes  

SciTech Connect

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.

Latchoumycandane, Calivarathan [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Seah, Quee Ming [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Tan, Rachel C.H. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Sattabongkot, Jetsumon [Armed Forces Research Institute of Medical Sciences, Bangkok 10400 (Thailand); Beerheide, Walter [Siam Life Science Ltd., Bangkok 10500 (Thailand); Boelsterli, Urs A. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore) and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117597 (Singapore)]. E-mail: phcbua@nus.edu.sg

2006-11-15

19

Role of Human CD36 in Bacterial Recognition, Phagocytosis and Pathogen-Induced C-Jun N-Terminal Kinase (JNK) - Mediated Signaling 1  

PubMed Central

Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6-signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacteria, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both Gram-positive and Gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36-transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to Gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with Gram-positive bacteria and lipotechoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40%–50% in human fibroblasts induced by both Gram-negative and Gram-positive bacteria as well as LPS. Of all MAP kinase signaling cascade inhibitors tested, only the inhibitor of JNK, a stress activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-?B inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an ?-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by Gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner. PMID:18981136

Baranova, Irina N.; Kurlander, Roger; Bocharov, Alexander V.; Vishnyakova, Tatyana G.; Chen, Zhigang; Remaley, Alan T.; Csako, Gyorgy; Patterson, Amy P.; Eggerman, Thomas L.

2013-01-01

20

Recent Advances in the Understanding of Interleukin2 Signal Transduction  

Microsoft Academic Search

Interleukin-2 is one of the critical cytokines that control the proliferation and differentiation of cells of the immune system. The present article briefly reviews the current and recently established knowledge on the intracellular signaling events that convert the initial interaction of IL-2 with its receptor into pathways leading to the various biological functions. A first step in IL-2 signaling is

Franck Gesbert; Maryvonnick Delespine-Carmagnat; Jacques Bertoglio

1998-01-01

21

Temporal sequence and cellular origin of interleukin-2 stimulated cytokine gene expression.  

PubMed Central

A study of activation of the cytokine network by interleukin 2, IL-2, may provide a rationale for devising cytokine combination and cytokine antagonist treatments with increased anti-tumour efficacy and decreased toxicity. We have investigated the expression of mRNA for 13 cytokines and three transcription factors during in vitro culture of peripheral blood mononuclear cells, PBMC, with IL-2. A consistent pattern of induction was seen in nine individuals, with early (2-24 h) induction of IL-1 beta, IL-6, tumour necrosis factor, TNF, lymphotoxin, LT, and gro. TNF and LT mRNA was expressed continually throughout culture, but levels of mRNA for IL-1 beta, IL-6, and gro declined by 24-48 h. After 48 h, PBMC began to express mRNA for IFN-gamma, IL-5, GM-CSF, and M-CSF. At 15 min to 1 h post IL-2 mRNA for c-fos, c-jun, and c-myc, and TNF was induced in three individuals studied. IL-4, IFN-alpha, and IL-1 alpha mRNA was not detected. Only a minority of cells expressed mRNA for TNF, IL-1 beta, IL-6 and IFN-gamma, and monocytes were the main source. Levels of cytokine protein in culture supernatants mirrored the pattern of mRNA induction. This in vitro model shows clear parallels with the reported in vivo production of cytokines during IL-2 therapy, and may prove useful in designing new therapeutic strategies. Images Figure 1 Figure 2 Figure 3 PMID:8439502

Saraya, K. A.; Balkwill, F. R.

1993-01-01

22

Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-?B Pathway: A Mouse Cardiomyocyte Model  

PubMed Central

Ginsenoside Rb3 is extracted from the plant Panax ginseng and plays important roles in cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. NF-?B is an important transcription factor involved in I/R injury. However, the underlying mechanism of ginsenoside Rb3 in myocardial I/R injury remains poorly understood. In the current study, a model of myocardial I/R injury was induced via oxygen and glucose deprivation (OGD) followed by reperfusion (OGD-Rep) in mouse cardiac myoblast H9c2 cells. Our data demonstrate that ginsenoside Rb3 suppresses OGD-Rep-induced cell apoptosis by the suppression of ROS generation. By detecting the NF-?B signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-?B activity. Ginsenoside Rb3 inhibits the upregulation of phospho-I?B-? and nuclear translocation of NF-?B subunit p65 which are induced by ORD-Rep injury. In addition, the extract also inhibits the OGD-Rep-induced increase in the expression of inflammation-related factors, such as IL-6, TNF-?, monocyte chemotactic protein-1 (MCP-1), MMP-2 and MMP-9. However, LPS treatment alleviates the protective roles of ginsenoside Rb3 and activates the NF-?B pathway. Finally, the upstream factors of NF-?B were analyzed, including the Akt/Foxo3a and MAPK signaling pathways. We find that ginsenoside Rb3 pretreatment only decreases the phosphorylation of JNK induced by OGD-Rep injury, an indicator of the MAPK pathway. Importantly, an inhibitor of phospho-JNK, SP600125, protects against OGD-Rep induced apoptosis and inhibited NF-?B signaling pathway, similar to the roles of ginsenoside Rb3. Taken together, our results demonstrate that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is attributed to the inhibition of JNK-mediated NF-?B activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury. PMID:25084093

Xie, Zulong; Yang, Shuang; Xu, Wei; Hou, Jingbo; Yu, Bo

2014-01-01

23

Metastatic Kidney Cancer: Treatment with Infusional Interleukin2 Plus Famotidine  

Microsoft Academic Search

\\u000a The administration of high dose continuous infusion Interleukin-2 is able to elicit cytolysis of cancer cells by lymphocytes,\\u000a predominantly CD56 positive natural killer cells. These Lymphokine Activated Killer cells (LAK) are able to lyse natural killer\\u000a cell-resistant tumor cells in vitro and renal cancer cells in vivo (Ellis et al. 1988; McMannis et al. 1988; Weil-Hillman\\u000a et al. 1989; Horton

Walter D. Y. Quan JR; Francine M. Quan

24

Serum soluble interleukin-2 receptor levels in familial Mediterranean fever  

Microsoft Academic Search

OBJECTIVE: To investigate serum soluble interleukin-2 receptor (sIL-2R) in familial Mediterranean fever (FMF) and assess its role in acute FMF crisis. METHODS: Serum sIL-2R concentrations were measured in patients with FMF during acute crises and during inactive periods of the disease, using an immunoenzymatic assay kit. Twenty four FMF patients during acute crisis (active FMF), 17 patients with inactive FMF,

E Erken; R Güne?açar; S Ozbek; K Konca

1996-01-01

25

Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene.  

PubMed Central

We demonstrate that purified interleukin 2 (IL-2) can directly upregulate IL-2 receptor expression on phytohemagglutinin-activated T lymphocytes maintained in culture until IL-2 receptor expression had markedly declined. The IL-2-induced increase in IL-2 receptor number is maximal within 12 hr, requires new RNA and protein synthesis, and is mediated by an interaction of ligand with the high-affinity receptors for IL-2. IL-2 stimulation results in increased accumulation of IL-2 receptor mRNA within 4 hr, while an increase in IL-2 receptor gene transcription is detected within 30 min in isolated nuclei. In addition, IL-2 incubation results in increased amounts of c-myc and transferrin receptor mRNA, but it does not augment levels of mRNA encoding the beta chain of the T-cell receptor for antigen. These results demonstrate that IL-2 can directly upregulate transcription and expression of its own receptor and, therefore, indicate that IL-2 may regulate IL-2-dependent immune responses, in part, by influencing the expression of IL-2 receptors. Images PMID:2987968

Depper, J M; Leonard, W J; Drogula, C; Krönke, M; Waldmann, T A; Greene, W C

1985-01-01

26

Interleukin-2 dependent cytotoxic T-cell clones  

SciTech Connect

A method is described of stimulating production of the lymphokines ..cap alpha..-interferon and ..beta..-interferon by interleukin-2 dependent cytotoxic cultured T-cell lines comprising administering to a T-cell line selected from the group consisting of T-cell lines CTLL-RP (CRL 8201), CTLL-R8 (CRL 8202), CTLL-R9 (CRL 8203), CTLL-R11 (CRL 8204), and CTLL-R12 (CRL 8205). An amount of an antigen selected from the group consists of Newcastle Disease Virus and Sendai Virus sufficient to cause stimulation of production of the lymphokines.

Palladino, M.

1987-07-28

27

Effect of spaceflight on lymphocyte proliferation and interleukin-2 production  

NASA Technical Reports Server (NTRS)

In this study, inguinal lymp node lymphocytes from rats flown on the Cosmos 2044 mission were tested for proliferation and interleukin-2 (IL-2) production. Cells cultured with mitogenic lectins, phorbol ester, and calcium ionophore, or T-cell mitogen and lymphokine, were assayed for DNA synthesis by (H-3) thymidine incorporation. Lymphocytes incubated with a T-cell mitogen alone also were tested for IL-2 production. Proliferation of lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from control and flown rats produced similar amounts of IL-23. Thus microgravity may act on lymphocytes in a tissue-specific manner, a new finding that could impact on the evaluation of spaceflight effects on immunocompetence.

Nash, Patricia V.; Konstantinova, Irina V.; Fuchs, Boris B.; Rakhmilevich, Alexandr L.; Lesniak, A. T.; Mastro, Andrea M.

1992-01-01

28

Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.  

PubMed

In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels. PMID:16549279

Avall-Jääskeläinen, Silja; Palva, Airi

2006-06-15

29

Differential regulation of transforming growth factor beta and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction  

Microsoft Academic Search

Summary The regulation of mKNA encoding transforming growth factor 3 (TGF-\\/3) and interleukin 2 (I1,2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-3 mRNA and IL-2 mRNA are regulated differentially in normal human T cells,

B. Li; P. K. Sehajpal; A. Khanna; H. Vlassara; A. Cerami; K. H. Stenzel; M. Suthanthiran

1991-01-01

30

DIFFERENTIAL EFFECTS OF HUMAN AND PORCINE INTERLEUKIN 2 ON NATURAL KILLING (NK) ACTIVITY OF NEWBORN PIGLETS  

E-print Network

DIFFERENTIAL EFFECTS OF HUMAN AND PORCINE INTERLEUKIN 2 ON NATURAL KILLING (NK) ACTIVITY OF NEWBORN HUMAINE ET PORCINE SUR L'ACTIVITÉ NK DES LYMPHOCYTES DE PORCELETS NOUVEAU-NÉS ET DE PORCS ADULTES. &horbar effets de l'Interleukine 2 (IL2) sur leur activité NK. Pour cela, de grandes quantités d'IL2 porcine ont

Paris-Sud XI, Université de

31

Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells.  

PubMed Central

Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients. Images PMID:8207793

Westendorp, M O; Li-Weber, M; Frank, R W; Krammer, P H

1994-01-01

32

Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease - An association study and expression analysis.  

PubMed

Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p?=?0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p?=?0.004 and p?=?0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p?=?0.031 and p?=?0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p?=?0.036) and positively with serum antibodies to 21OH (p?=?0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p?=?0.022) and soluble IL2Ra secretion (p?=?0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61?±?4.3 versus 1.71?±?3.2?pg/mL, p?interleukin-2 and sIL2Ra. PMID:25347332

Fichna, Marta; ?urawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Za?uska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy

2015-03-01

33

Intravenous ascorbic acid as an adjuvant to interleukin-2 immunotherapy  

PubMed Central

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. PMID:24884532

2014-01-01

34

Biosynthetic processing of human interleukin-2 receptor (Tac antigen).  

PubMed

Biosynthetic processing of the T-cell surface receptor for interleukin-2 was investigated in a cultured human T-cell line MT-1 by means of metabolic and cell surface radiolabeling followed by immunoprecipitation with a monoclonal anti-receptor antibody (anti-Tac) and analysis by one- and two-dimensional polyacrylamide gel electrophoresis. The nascent precursor of the receptor (Mr = about 40,000, pI = 6.2-6.5) underwent a post-translational modification giving rise to the mature receptor (IL-2R; Mr = 60,000-65,000, pI = 4.2-4.7) within 2-4 hr. The post-translational processing of IL-2R caused a 20,000-25,000 increase in apparent molecular weight and a 2.0-2.5 acidic shift in the isoelectric point. The increase in molecular weight was attributable mainly to addition of sugar residues including glucosamine and galactose, and the charge shift to the addition of sialic acids. A carboxylic ionophore monensin completely blocked the maturation of IL-2R at the mid-stage of the processing. Fatty acid attachment appeared to comprise one of the steps of the post-translational modification. Two-dimensional analyses of IL-2R biosynthesis enabled identification of the precursor of IL-2R and its intermediate forms, from which it was partially possible to estimate reactions involved in the maturation of the precursor molecule. PMID:3929032

Wano, Y; Uchiyama, T; Yodoi, J; Uchino, H

1985-01-01

35

Effect of isoprinosine on sialylation of interleukin-2.  

PubMed

The effects of Isoprinosine (ISO) on interleukin-2 (IL-2) production by human peripheral blood mononuclear cells (PBMC) were investigated. Treatment (of human PBMC) with ISO enhanced IL-2 production by PBMC from 7 of 10 normal individuals. However, no augmentation of IL-2 production was observed when cultures of HUT-78 cells, a human leukemic T cell line, were treated with ISO. IL-2 purified from supernatants of human PBMC treated with ISO exhibited pI values of 5.5 and 6.4. IL-2 prepared from untreated PBMC exhibited a single pI value of 8.2. The pI value of IL-2 prepared from ISO-treated PBMC shifted to 8.2 after treatment with neuraminidase, demonstrating that the IL-2 molecules isolated from ISO-treated PBMC possessed sialic acid. The pI values of the IL-2 isolated from ISO-treated and untreated HUT-78 culture supernatants were identical (pI = 7.8) and were not modified by neuraminidase treatment. These results suggest that the increase in IL-2 production following treatment of PBMC with ISO may be mediated through the activation of a distinct subset of IL-2 producing cells. Furthermore, the sialylation of IL-2 may be of physiologic and immunopharmacologic importance. PMID:2424829

Tsang, K Y; Boutin, B; Pathak, S K; Donnelly, R; Koopmann, W R; Fleck, R; Miribel, L; Arnaud, P

1986-04-01

36

Expression of interleukin-2 receptor on T cell chronic lymphocytic leukemia cells and their response to interleukin-2.  

PubMed

We analyzed peripheral blood leukemic cells from six patients with T cell chronic lymphocytic leukemia (T-CLL) with monoclonal antibodies including the anti-Tac antibody, which recognizes the receptor for interleukin 2 (IL 2). The patients were divided into two groups according to the reactivity of the monoclonal antibodies. Leukemic cells from three patients with T-CLL reacted with OKT3 and T4 but not T8, whereas those from the remaining three patients reacted with OKT3 and T8 but not T4. IL 2 receptor, which is expressed on activated T cells but not on resting T cells, was preferentially expressed on T4+ T-CLL cells. The IL 2 receptor on T4+ T-CLL cells was indistinguishable from that on normal activated T cells with respect to molecular weight and downregulation by the anti-Tac antibody. Moreover, fresh T4+ T-CLL cells, but not T8+ T-CLL cells, proliferated in response to exogenous IL 2 without prior activation, and this proliferation was inhibited by the anti-Tac antibody. These results suggest that malignant growth of T4+ T-CLL cells can be regulated by IL 2 not only in vitro but also in vivo. PMID:3080037

Tsudo, M; Uchiyama, T; Umadome, H; Wano, Y; Hori, T; Tamori, S; Uchino, H; Kita, K; Chiba, S; Mitsutani, S

1986-02-01

37

Continuous release of interleukin-2 from liposomal IL-2 (mixture of interleukin-2 and liposomes) after subcutaneous administration to mice.  

PubMed

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small and hydrophobic liposomes by simple mixing under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 displayed better distribution after intravenous administration in mice and improved therapeutic effect against experimental M5076 metastases, as reported previously. In this study, the elimination of IL-2 from the dosing area was investigated when the liposomal IL-2 was administered to mice subcutaneously. The results suggest that the release of IL-2 from this liposome was continuous and almost complete. The mean residence time (MRT) of IL-2 in the dosing area was 11.0 +/- 1.65 hr. This resulted in the 8-fold times enhancement of MRT in the systemic circulation by the presence of liposomes, and IL-2 was detected in the serum for 2 days. Using this liposomal IL-2 is expected to have the potential to decrease the number of injections and enhance the efficacy of IL-2 in immunotherapies and therapies against tumor. PMID:14677775

Kanaoka, Eri; Takahashi, Kouji; Yoshikawa, Takayoshi; Jizomoto, Hiroaki; Nishihara, Yoshitaka; Hirano, Koichiro

2003-11-01

38

Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.  

PubMed

Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast. PMID:8769948

Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

1996-02-15

39

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors.  

PubMed

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen. PMID:3108674

Dukovich, M; Wano, Y; Le thi Bich Thuy; Katz, P; Cullen, B R; Kehrl, J H; Greene, W C

40

Decreased production of interleukin-2 receptors after immunotherapy to house dust  

Microsoft Academic Search

The magnitude of the T-cell immune response depends on both the amount and the duration of interaction between interleukin 2 (IL-2) and interleukin-2 receptors (IL-2R). In a previous study, we found that IL-2 production was decreased after immunotherapy. In order to delineate further the mechanisms for the decreased lymphoproliferative response after immunotherapy,in vitro production of soluble IL-2R (SIL-2R) was studied

Kue-Hsiung Hsieh

1988-01-01

41

Arf6-GEF BRAG1 regulates JNK-mediated synaptic removal of GluA1-containing AMPA receptors: a new mechanism for nonsyndromic X-linked mental disorder  

PubMed Central

Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disability. BRAG1 is a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif, however the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors (-Rs), depresses AMPA-R-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic X-linked intellectual disability. PMID:22915114

Myers, Kenneth R.; Wang, Guangfu; Sheng, Yanghui; Conger, Kathryn K.; Casanova, James E.; Zhu, J. Julius

2012-01-01

42

Soluble interleukin-2 receptor in stage I-III melanoma.  

PubMed

The aim of the present study was to assess the prognostic value of soluble interleukin-2 receptor (sIL-2R) serum levels in stage I-III melanoma patients. The levels of sIL-2R were determined using an enzyme immunometric test kit in 329 patients affected by malignant melanoma (MM) from 1995 to 2004. Correlations between sIL-2R values, baseline patients and tumour features were studied by contingency tables and the chi-square test. The Kaplan-Meier product limit method was applied to plot disease-free survival (DFS) curves. Univariate analysis was performed with the Log-rank test. Cox proportional-hazards regression was used to analyse the effect of several risk factors on DFS. In total, 2330 blood samples were collected during follow-up of 329 MM patients. Forty-five (13.7%) patients had Breslow tumour thickness1.00 and 2.00 and 4.00 mm. Ulceration was present in 64 cases (19.4%). Thirty-nine sentinel lymph nodes (SLNs) (11.8%) were infiltrated by MM. Soluble IL-2R values ranged from 130 to 1420 U/ml; median value was 500 U/ml. One hundred twenty-one (36.8%) patients presented with sIL-2R>600 U/ml at first measure (FM), 194 patients (58.9%) with values increasing up to or more than 600 U/ml [increasing values (IV) pattern]. A correlation was found between Breslow's tumour values and the IV sIL-2R pattern group (P=0.0304 with chi2 test). Gender, presence of ulceration, Breslow tumour thickness, FM and IV sIL-2R pattern groups had a significant prognostic value for DFS. At multivariate analysis, presence of ulceration, gender, FM and IV sIL-2R pattern groups emerged as independent prognostic factors for DFS. The 5-year DFS rate was 88% for patients with FM<600 U/ml and 76.9% for patients with FM>600 U/ml. In IV pattern, the 5-year DFS rate was 69.5% compared to 87% for patients with no sIL-2R values>600 U/ml during follow-up. sIL-2R values are associated with progression of MM. Further studies are needed to address the role of the IL-2/IL-2R/sIL-2R axis in melanoma biology. PMID:16517174

Ottaiano, Alessandro; Leonardi, Enrico; Simeone, Ester; Ascierto, Paolo Antonio; Scala, Stefania; Calemma, Rosa; Bryce, Jane; Caracò, Corrado; Satriano, Rocco A; Gianfranco, Nicoletti; Franco, Renato; Botti, Gerardo; Castello, Giuseppe

2006-02-01

43

Chemical Modification of Recombinant Interleukin 2 by Polyethylene Glycol Increases Its Potency in the Murine Meth A Sarcoma Model  

Microsoft Academic Search

Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life. This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol. The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate\\/polyacrylamide gel electrophoresis and isoelectric focusing. This conjugate was compared to unmodified

Nandini V. Katre; Michael J. Knauf; Walter J. Laird

1987-01-01

44

Activation of Interleukin 2 and Interleukin 2 Receptor (Tac) Promoter Expression by the Trans-Activator (tat) Gene Product of Human T-Cell Leukemia Virus, Type I  

Microsoft Academic Search

Cotransfection of cDNA encoding the transactivator gene product of human T-cell leukemia virus, type I (HTLV-I) (tat-I), which acts in trans to augment viral gene expression, has revealed strong regulatory effects of this viral protein on the inducible cellular promoters governing human interleukin 2 (IL-2) and IL-2 receptor (Tac) gene expression. The tat-I protein stimulates a 3- to 6-fold increase

M. Siekevitz; M. B. Feinberg; N. Holbrook; F. Wong-Staal; W. C. Greene

1987-01-01

45

Phorbol ester replacement of helper cell and interleukin 2 requirements in gamma interferon production.  

PubMed Central

Immune interferon (IFN-gamma) production by mitogen-stimulated C57BL/6 mouse spleen cells required both Lyt 1+,2- and Lyt 1-,2+ lymphocytes. The Lyt 1-,2+ cells were the IFN-gamma producers, whereas the Lyt 1+,2- cells were the helpers. Interleukin 2 mediated the Lyt 1+,2- cell help. Phorbol myristic acetate (10 ng/ml) completely replaced Lyt 1+,2- helper cell or interleukin 2 requirements in IFN-gamma production. The phorbol ester exerted its helper effects by direct interaction with IFN-gamma-producing Lyt 1-,2+ cells and not through stimulation of production of interleukin 2 by Lyt 1+, 2- cells. Suppressor cell effects in IFN-gamma production were also completely abrogated by phorbol myristic acetate. The data suggest a mechanism for phorbol myristic acetate regulation of IFN-gamma production at the cellular level. PMID:6178691

Johnson, H M; Torres, B A

1982-01-01

46

In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey  

NASA Technical Reports Server (NTRS)

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

1996-01-01

47

IDENTIFICATION OF A T CELL-DERIVED B CELL GROWTH FACTOR DISTINCT FROM INTERLEUKIN 2  

Microsoft Academic Search

The importance of murine monokines and lymphokines in T cell immune responses has been emphasized by the development of T cell cloning technology (1) and the identification of cloned tumor lines that respond to or secrete these factors (2, 3). Such studies have shown that the monokine interleukin 1 (IL-1) 1 induces certain T cells to secrete interleukin 2 (IL-2),

MAUREEN HOWARD; JOHN FARRAR; MARY HILFIKER; BARBARA JOHNSON; KIYOSHI TAKATSU; TOSHIYUKI HAMAOKA; WILLIAM E. PAUL

48

Toxicity and Immunomodulatory Effects of Interleukin2 After Autologous Bone Marrow Transplantation for Hematologic Malignancies  

Microsoft Academic Search

HE SUCCESS OF autologous bone marrow transplan- T tation (ABMT) for disseminated hematologic malig- nancies is limited largely by the high incidence of recur- rence of the malignancy after ABMT.'.' The recurrences are attributed to the failure of the chemoradiotherapy to eradicate all residual disease and, possibly, to the out- growth of tumor cells contaminating the infused autologous marrow. Interleukin-2

Carl M. Higuchi; John A. Thompson; Finn B. Petersen; C. Dean Buckner; Alexander Fefer

1991-01-01

49

Prolonged Interleukin-2Ra Expression on Virus-Specific CD8+  

E-print Network

; Williams and Bevan, 2007). Moreover, the onset and kinetics of contraction and memory CD8+ T cell fate cytokines, and growth factors are implicated in regulating T cell fate decisions (Ahmed and Rouse, 2006Immunity Article Prolonged Interleukin-2Ra Expression on Virus-Specific CD8+ T Cells Favors

50

Murine interleukin 2 receptor beta chain: dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion.  

PubMed Central

The functional, high-affinity interleukin 2 receptor (IL-2R) consists of at least two receptor components, IL-2R alpha (p55) and IL-2R beta (p70-75). The cDNA encoding the murine IL-2R beta has been isolated by using the previously cloned cDNA for human IL-2R beta as a probe. Analysis of the cDNA revealed that the murine IL-2R beta shows a marked homology with the human IL-2R beta and that it is also structurally related to other cytokine receptors such as erythropoietin receptor. The cDNA-directed murine IL-2R beta formed high-affinity IL-2R in conjunction with the endogenous IL-2R alpha in a murine pro-B-cell line and could transduce IL-2-induced growth signal. In mouse lymphoma line EL-4, the IL-2R beta gene was found to be rearranged by the insertion of the long terminal repeat sequence of an intracisternal A particle, giving rise to constitutive expression of the IL-2R beta mRNA. Images PMID:2155425

Kono, T; Doi, T; Yamada, G; Hatakeyama, M; Minamoto, S; Tsudo, M; Miyasaka, M; Miyata, T; Taniguchi, T

1990-01-01

51

Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene  

Microsoft Academic Search

Inflammatory lesions of the skin such as erythema, depigmentation and hair loss were observed in C57\\/BL6(B6) transgenic mice that carried an intact human genomic interleukin-2 gene (gIL-2 transgenic mice). Accumulation of T lymphocytes in the perivascular and periadnexal areas of the dermis was the first change, followed by dermal papillary oedema, which occurred before the development of macroscopic skin lesions.

M. Akiyama; M. Yokoyama; M. Katsuki; S. Habu; T. Nishikawa

1993-01-01

52

RESPONSE OF RESTING HUMAN PERIPHERAL BLOOD NATURAL KILLER CELLS TO INTERLEUKIN 2  

Microsoft Academic Search

Resting T cells can be activated to proliferate by antigens and mitogenic lectins, but their proliferation depends on a hormone-like lymphokine, T cell growth factor or interleukin 2 (IL-2) ~ (1-2). Activation of T cells by a mitogenic stimulus induces both secretion of minute quantities of IL-2 and the appearance of a high-affinity receptor for IL-2 on the T cell

GIORGIO TRINCHIERI; MICHIKO MATSUMOTO-KOBAYASHI; STEVEN C. CLARK; JASBIR SEEHRA; LUCILLE LONDON; BICE PERUSSIA

53

Evolution of the levels of soluble interleukin-2 receptors during Plasmodium falciparum and P. vivax malaria.  

PubMed Central

Increased levels of soluble interleukin-2 receptors (IL-2R) in serum were observed in both Plasmodium falciparum- and P. vivax-infected individuals compared with nonparasitemic subjects. Clinical symptoms of P. falciparum malaria were associated with higher levels of soluble IL-2R. Temporal evolution in serum of IL-2R during the course of a malaria attack mimicked the kinetics of soluble IL-2R under experimental conditions. PMID:2671038

Deloron, P; Lepers, J P; Coulanges, P

1989-01-01

54

Interleukin 2 Receptors and Detergent-Resistant Membrane Domains Define a Clathrin-Independent Endocytic Pathway  

Microsoft Academic Search

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes

Christophe Lamaze; Annick Dujeancourt; Takeshi Baba; Charles G. Lo; Alexandre Benmerah; Alice Dautry-Varsat

2001-01-01

55

Antitumor Efficacy of Recombinant Human Interleukin2 Combined with Sorafenib Against Mouse Renal Cell Carcinoma  

Microsoft Academic Search

Objective: Recombinant human interleukin-2 (rhIL-2) has been clinically used in the treatment of renal cell carcinoma (RCC). Sorafenib, a multi-targeted kinase inhibitor, has been approved for RCC as well as IL-2. The purpose of this study was to evaluate the antitu- mor efficacy of IL-2 combined with sorafenib in three different murine renal cancer models using Renca cells. Methods: We

Motofumi Iguchi; Mitsunobu Matsumoto; Kanji Hojo; Toru Wada; Yoshiyuki Matsuo; Akinori Arimura; Kenji Abe

56

Phosphatidylinositol 3Kinase Couples the Interleukin2 Receptor to the Cell Cycle Regulator E2F  

Microsoft Academic Search

Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5

Paul Brennan; Jane W Babbage; Boudewijn M. T Burgering; Bernd Groner; Karin Reif; Doreen A Cantrell

1997-01-01

57

The Role of Interleukin2 in Combination Adenovirus Gene Therapy for Head and Neck Cancer  

Microsoft Academic Search

Interleukin-2 (IL-2) gene therapy alone and in com- bination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcino- mas in C3H\\/HeJ mice were directly injected with recombinant adenoviral vectors carrying both ther- apeutic and control genes. One week after adeno- viral

Bert W. O'Malley; Duane A. Sewell; Daqing Li; Ken-ichiro Kosai; Shu-Hsia Chen; Savio L. C. Woo; Ling Duan

1997-01-01

58

Alteration of dacarbazine pharmacokinetics after interleukin-2 administration in melanoma patients  

Microsoft Academic Search

In an effort to improve the treatment of metastatic malignant melanoma, we evaluated the sequential administration of the chemotherapeutic agent dacarbazine (DTIC) and the biological response modifier interleukin-2 (rIL-2) in a phase I–II study. Since the combination of biological response modifiers and chemotherapeutic agents could alter drug disposition, we evaluated the pharmacokinetics of DTIC and its major metabolite, 5-aminoimidazole 4-carboxamide

Guy G. Chabot; Lawrence E. Flaherty; Manuel Valdivieso; Laurence H. Baker

1990-01-01

59

Acute Dyslipoproteinemia Induced by Interleukin2: Lecithin:Cholesteryl Acyltransferase, Lipoprotein Lipase, and Hepatic Lipase Deficiencies  

Microsoft Academic Search

Recombinant human interleukin-2 (rIL-2) is used to treat refrac- tory cancers. During such treatment, patients develop severe hypo- cholesterolemia along with striking alterations in the concentration and composition of the circulating lipoproteins. The present study was undertaken to gather information about the pathogenesis of these abnormalities. Patients were studied before-, during- and after a 5-day course of high dose iv

LINDA K. KWONG; DAVID N. RIDINGER; MICHAEL BANDHAUER; JOHN H. WARD; WOLFRAM E. SAMLOWSKI; PER-HENRIK IVERIUS; HAYDN PRITCHARD; DANA E. WILSON

2010-01-01

60

Reduced incidence of hepatic metastases by perioperative treatment with recombinant human interleukin-2  

Microsoft Academic Search

Abdominal operations induce immunosuppression during the time when tumors are manipulated and tumor cells are released into\\u000a the circulation. The authors tested the hypothesis that the combined effect of these factors may promote the development of\\u000a metastatic tumor implants and that perioperative treatment with Human Recombinant Interleukin-2 (RIL-2), a known immunostimulant\\u000a oft, natural killer (NK), and lymphokine activated killer (LAK)

James L. Weese; Sherrie E. Emoto; Paul M. Sondel

1987-01-01

61

Differential Regulation of Colony-Stimulating Factors and Interleukin 2 Production by Cyclosporin A  

Microsoft Academic Search

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma -interferon, its effect on the production of granulocyte\\/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study,

M. Bickel; H. Tsuda; P. Amstad; V. Evequoz; S. E. Mergenhagen; S. M. Wahl; D. H. Pluznik

1987-01-01

62

Inhibition of Stimulated Interleukin2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect  

Microsoft Academic Search

Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, mea- surement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic. Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated

C. Michael Stein; John J. Murray; Alastair J. J. Wood

63

Interleukin 2 production in a family with systemic lupus erythematosus and a C4Q0 heterozygous inheritance.  

PubMed Central

Interleukin 2 production was studied in a family with systemic lupus erythematosus (SLE) and a C4Q0 heterozygous inheritance. Autoimmune manifestations seemed to be associated with the HLA haplotype containing the C4Q0 allele, which was shared by all four ill family members. Concentrations of interleukin 2, however, did not associate either with the haplotype or with the clinical or serological manifestations, as diminished concentrations of interleukin 2 were found in only two subjects with SLE. Thus the defect in this family seemed to be acquired rather than genetically conditioned. PMID:1888202

Gutierrez, C; Cabrero, E; Vicario, J L; Martín Villa, M; Rengel, M A; Gomez Campdera, F J; Yebra, M; Fernández-Cruz, E; Arnaiz Villena, A

1991-01-01

64

Partial restoration of impaired interleukin-2 production and Tac antigen (putative interleukin-2 receptor) expression in patients with acquired immune deficiency syndrome by isoprinosine treatment in vitro.  

PubMed Central

The in vitro effects of isoprinosine (ISO) on interleukin-2 (IL-2) production, the expression of Tac antigen (IL-2 receptor) on lymphocytes, and the ability of Leu 3(+) cells to absorb interleukin-1 (IL-1) were investigated in 10 patients with acquired immune deficiency syndrome (AIDS). In 9 of the 10 patients, production of IL-2 from mononuclear cells and Leu 3(+) cells was depressed; expression of Tac antigen on mononuclear cells and Leu 2(+) cells was found to be depressed in 9 of 10 patients. The ability of the Leu 3(+) lymphocytes to absorb IL-1 was depressed in all (four of four) patients studied. After ISO treatment, IL-2 production, Tac antigen expression and IL-1 absorption were restored to normal or near normal levels in most of the patients. These results suggest that ISO has an immunostimulating capacity in AIDS patients and that the potential of ISO in immune response restoration in AIDS patients deserves critical consideration. PMID:2581997

Tsang, K Y; Fudenberg, H H; Galbraith, G M; Donnelly, R P; Bishop, L R; Koopmann, W R

1985-01-01

65

Distribution of interleukin-2, -4, -10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus.  

PubMed

In the present study, MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with 35S-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta 1 were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (TH1) and TH2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. PMID:10401528

Simark-Mattsson, C; Bergenholtz, G; Jontell, M; Eklund, C; Seymour, G J; Sugerman, P B; Savage, N W; Dahlgren, U I

1999-06-01

66

The effect of protein deficiency and M. bovis BCG vaccination on interleukin 2 activity in tuberculous ginea pigs  

E-print Network

THE EFFECT OF PROTEIN DEFICIENCY AND M. bovis BCG VACCINATION ON INTERLEUKIN 2 ACTIVITY IN TUBERCULOUS GUINEA PIGS A THESIS by REBECCA LYNNE PARR Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment... of the requirements for the degree of MASTER OF SCIENCE December 1987 Major subject: Veterinary Microbiology THE EFFECT OF PROTEIN DEFICIENCY AND M~ BOVIS BCG VACCINATION ON INTERLEUKIN 2 ACTIVITY IN TUBERCULOUS GUINEA PIGS A Thesis by REBECCA LYNNE PARR...

Parr, Rebecca Lynne

1987-01-01

67

Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy.  

PubMed

Recombinant Newcastle disease virus (rNDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) delivered by rNDV. We demonstrated that rNDV expressing TRAIL (rNDV-TRAIL) or both human IL-2 and TRAIL (rNDV-IL-2-TRAIL) significantly enhanced inherent anti-neoplastic of rNDV by inducing apoptosis. And we showed that apoptosis-related genes mRNA expression was increased after treated with rNDV-TRAIL or rNDV-IL-2-TRAIL compared with rNDV and rNDV-IL-2. We also demonstrated that both rNDV-IL-2 and rNDV-IL-2-TRAIL induced proliferation of the CD4(+) and CD8(+) in treated mice and elicited expression of TNF-? and IFN-? antitumor cytokines. These mice treated with oncolytic agents exhibited significant reduction in tumor development compared with mice treated with the parental virus. In addition, experiments in both hepatocellular carcinoma and melanoma-bearing mice demonstrated that the genetically engineered rNDV-IL-2-TRAIL exhibited prolonged animals' survival compared with rNDV, rNDV-IL-2, and rNDV-TRAIL. In conclusion, the immunotherapy and oncolytic virotherapy properties of NDV can be enhanced by the introduction of IL-2 and TRAIL genes, whose products initiated a broad cascade of immunological affects and induced tumor cells apoptosis in the microenvironment of the immune system. PMID:24971746

Bai, Fu-Liang; Yu, Yin-Hang; Tian, Hui; Ren, Gui-Ping; Wang, Hui; Zhou, Bing; Han, Xiao-Hui; Yu, Qing-Zhong; Li, De-Shan

2014-09-01

68

Inhibition of G-Protein ?? Signaling Enhances T Cell Receptor-Stimulated Interleukin 2 Transcription in CD4+ T Helper Cells  

PubMed Central

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein ?? (G??) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of G??, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. G?1 and G?2 mRNA accounted for >99% of G? mRNA, and small interfering RNA (siRNA)-mediated silencing of G?1 but not G?2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking G?? enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking G?? also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous G?? inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking G?? in CD4+ T helper cells could have applications for autoimmune diseases. PMID:25629163

Yost, Evan A.; Hynes, Thomas R.; Hartle, Cassandra M.; Ott, Braden J.; Berlot, Catherine H.

2015-01-01

69

Co-immunotherapy with interleukin-2 and taurolidine for progressive metastatic melanoma  

Microsoft Academic Search

Background  Recombinant interleukin-2(rIL-2) therapy in metastatic melanoma is limited by toxicities, particularly vascular leak syndrome(VLS).\\u000a Taurolidine potentiates the anti-neoplastic effects of IL-2 while reducing its associated endothelial cell dysfunction in\\u000a experimental settings. We hypothesized that co-administration of rIL-2 with taurolidine could enhance tolerability without\\u000a weakening effectiveness.\\u000a \\u000a \\u000a \\u000a Methods  Eleven patients with progressive metastatic melanoma received high-dose rIL-2 with co-infusion of taurolidine. Patients were

G. C. O’Brien; R. A. Cahill; D. J. Bouchier-Hayes; H. P. Redmond

2006-01-01

70

Metastatic type-2 papillary renal cell carcinoma responded to interleukin-2 therapy: case report  

Microsoft Academic Search

This report documents a case of metastatic papillary renal cell carcinoma (PRCC) which successfully responded to interleukin-2\\u000a (IL-2) therapy. A 59-year-old male presented with a left renal mass measuring 3.0 cm in diameter and a right adrenal mass\\u000a measuring 5.0 cm in diameter. He underwent a left partial nephrectomy and a right adrenalectomy. The histological findings\\u000a revealed pT1bN1M1 type-2 PRCC and metastatic

Takeo Kosaka; Shuji Mikami; Akira Miyajima; Mototsugu Oya

2008-01-01

71

Interleukin 2-dependent release of interleukin 3 activity by T4+ human T-cell clones.  

PubMed Central

We have isolated and studied two alloreactive, T4+, human lymphocyte clones that release interleukin 2 (IL-2) and interleukin 3 (IL-3) bioactivities upon stimulation with IL-2, alloantigen, or Sepharose-conjugated antibodies directed against the T3 protein. Anti-IL-2 receptor monoclonal antibodies block IL-2-, alloantigen-, or anti-T3-stimulated IL-3 release. Hence, release of IL-3 activity in each circumstance is rigorously dependent upon activation of the IL-2 receptor. Even low, nonmitogenic concentrations of recombinant IL-2 stimulated IL-3 release. PMID:2413452

Ythier, A A; Abbud-Filho, M; Williams, J M; Loertscher, R; Schuster, M W; Nowill, A; Hansen, J A; Maltezos, D; Strom, T B

1985-01-01

72

Ability of isoprinosine to restore interleukin-2 production and T cell proliferation in autoimmune mice.  

PubMed Central

Autoimmune mice bearing the single autosomal recessive gene 1pr are unable to produce the T cell growth factor, interleukin-2 (IL-2). A physiological consequence of this defect is the inability of T cells from C57B1/6J-lpr/lpr mice to respond to antigen presented by macrophages. In an attempt to reverse these abnormalities, we administered the inosine containing drug isoprinosine. Injection of isoprinosine after antigen immunization restored both antigen presentation and IL-2 production. PMID:2412742

Fischbach, M; Talal, N

1985-01-01

73

Interleukin2 receptor ?-chain mutations in severe combined immunodeficiency with B-lymphocytes  

Microsoft Academic Search

Abstract  Severe combined immuno-deficiency (SCID) with a normal number of B-lymphocytes usually demonstrates an X-linked inheritance\\u000a and now is regarded as an interleukin-2-receptor (IL-2R) ?-chain gene defect. Here, we report the characterization of mutations\\u000a in the IL-2R ?-chain gene of six unrelated SCID patients. One large deletion, one short deletion, one nonsense mutation and\\u000a three single missense mutations were identified. The

I. Tsuge; H. Matsuoka; T. Abe; Y. Kamachi; S. Torii

1996-01-01

74

A chimera of interleukin 2 and a binding variant of aerolysin is selectively toxic to cells displaying the interleukin 2 receptor.  

PubMed

Aerolysin is a bacterial toxin that binds to glycosylphosphatidylinositol-anchored proteins (GPI-AP) on mammalian cells and oligomerizes, inserting into the target membranes and forming channels that cause cell death. We have made a variant of aerolysin, R336A, that has greatly reduced the ability to bind to GPI-AP, and as a result it is only very weakly active. Fusion of interleukin 2 (IL2) to the N terminus of R336A-aerolysin results in a hybrid that has little or no activity against cells that do not have an IL2 receptor because it cannot bind to the GPI-AP on the cells. Strikingly, the presence of the IL2 moiety allows this hybrid to bind to cells displaying high affinity IL2 receptors. Once bound, the hybrid molecules form insertion-competent oligomers. Cell death occurs at picomolar concentrations of the hybrid, whereas the same cells are insensitive to much higher concentrations of R336A-aerolysin lacking the IL2 domain. The targeted channel-forming hybrid protein may have important advantages as a therapeutic agent. PMID:17981806

Osusky, Milan; Teschke, Lisa; Wang, Xiaoying; Wong, Kevin; Buckley, J Thomas

2008-01-18

75

Serum soluble interleukin-2 receptor level as a prognostic indicator in gastric cancer.  

PubMed Central

T lymphocytes, activated by interleukin 2 during an anti-tumour response, release soluble interleukin 2 receptors (sIL-2R) into the bloodstream. We analysed the prognostic value of the serum sIL-2R level in gastric cancer. Serum concentration of sIL-2R in 96 gastric cancer patients and 100 healthy control subjects' was measured by enzyme-linked immunosorbent assay. All survivors were followed for more than 50 months. Serum sIL-2R level was considered with respect to prognosis, clinicopathological factors, other tumour markers and peripheral blood cell count. Stage III and IV patients had significantly higher sIL-2R levels than lower stage patients and control subjects. Stage III and IV gastric cancer patients were divided into 'high' and 'low' slL-2R groups based upon the control subjects' serum sIL-2R mean value plus one standard deviation. The high group had a significantly worse prognosis than the low group, although clinicopathological features and treatments were similar. Multivariate analysis demonstrated that the serum sIL-2R level is an independent indicator. The sIL-2R level did not correlate with carbohydrate antigen 19-9, however it did correlate with carcinoembryonic antigen (r = 0.22) and with numbers of peripheral blood monocytes (r = 0.54). In conclusion, serum sIL-2R may predict the outcome of gastric cancer patients with stage III or IV disease. PMID:9667652

Nakata, B.; Chung, K. H.; Kato, Y.; Yamashita, Y.; Inui, A.; Arimoto, Y.; Maeda, K.; Onoda, N.; Sawada, T.; Sowa, M.

1998-01-01

76

CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation  

PubMed Central

Lymphocyte recognition of antigen by the antigen-specific T cell receptor (TCR) and coreceptor complexes rapidly alters the cell's adhesive properties facilitating high avidity cell-ligand interactions necessary for lymphocyte development and function. Here, we report the expression of CD81 (target of antiproliferative antigen [TAPA]-1) on human thymocytes and the physical association of CD81 with CD4 and CD8 T cell coreceptors. Antibody ligation of CD81 on thymocytes promotes the rapid induction of integrin-mediated cell-cell adhesion via lymphocyte function-associated molecule-1 (LFA-1). Cross-linking CD81 is also shown to be costimulatory with signaling through the TCR/CD3 complex inducing interleukin 2-dependent thymocyte proliferation. These data suggest that a CD81-mediated pathway in thymocytes is involved in the regulation of both cell adhesion and activation. PMID:8920895

1996-01-01

77

Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody  

SciTech Connect

Administration of a rat monoclonal antibody (M7/20) directed against the murine interleukin 2 (IL 2) receptor in combination with sublethal x-irradiation of the recipient significantly enhanced the survival of skin allografts, both when the grafts were MHC disparate from the hosts and when only minor histocompatibility differences were present compared with untreated controls. No prolongation in graft survival was seen with either treatment alone at the dose employed. M7/20 and x-ray-treated allograft recipients also displayed significantly decreased alloantigen-specific reactivity against donor-strain spleen cells in both delayed-type hypersensitivity and cytotoxicity assays. Thus, such combination treatment reduces expression of host immune reactivity against graft determinants by several criteria. This work provides additional evidence that monoclonal antibodies directed against the IL 2 receptor may be useful in clinical transplantation.

Granstein, R.D.; Goulston, C.; Gaulton, G.N.

1986-02-01

78

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity.  

PubMed

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4(+) CD25(+) regulatory T cells, which are critical for maintaining immune homeostasis. PMID:17277778

Yamanouchi, Jun; Rainbow, Dan; Serra, Pau; Howlett, Sarah; Hunter, Kara; Garner, Valerie E S; Gonzalez-Munoz, Andrea; Clark, Jan; Veijola, Riitta; Cubbon, Rose; Chen, Show-Ling; Rosa, Raymond; Cumiskey, Anne Marie; Serreze, David V; Gregory, Simon; Rogers, Jane; Lyons, Paul A; Healy, Barry; Smink, Luc J; Todd, John A; Peterson, Laurence B; Wicker, Linda S; Santamaria, Pere

2007-03-01

79

Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp. lactis.  

PubMed

Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system. The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium. Sequence analysis revealed correct processing at the first amino acid of the mature protein. A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source. PMID:7747977

Steidler, L; Wells, J M; Raeymaekers, A; Vandekerckhove, J; Fiers, W; Remaut, E

1995-04-01

80

Interleukin-2 at the Crossroads of Effector Responses, Tolerance, and Immunotherapy  

PubMed Central

Interleukin-2 is a pleiotropic cytokine produced after antigen activation that plays pivotal roles in the immune response. Discovered as a T-cell growth factor, IL-2 additionally promotes CD8+ T cell and NK cell cytolytic activity, and modulates T cell differentiation programs in response to antigen, promoting naïve CD4+ T cell differentiation into T helper-1 (Th1) and T helper-2 (Th2) cells while inhibiting T helper-17 (Th17) and T follicular helper (Tfh) cell differentiation. Moreover, IL-2 is essential for the development and maintenance of T regulatory (Treg) cells and for activation-induced cell death, thereby mediating tolerance and limiting inappropriate immune reactions. In this review, we focus on the molecular mechanisms and complex cellular actions of IL-2, its cooperative and opposing effects with other cytokines, and how both promoting and blocking the actions of IL-2 are being utilized in clinical medicine. PMID:23352221

Liao, Wei; Lin, Jian-Xin; Leonard, Warren J.

2013-01-01

81

Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy  

Technology Transfer Automated Retrieval System (TEKTRAN)

Recombinant Newcastle disease virus (rNDV) has shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tu...

82

Soluble Interleukin 2 Receptor Levels, Temperament and Character in Formerly Depressed Suicide Attempters Compared with Normal Controls  

ERIC Educational Resources Information Center

An imbalance of the immune system and mixed personality profiles in suicide attempters have been reported. As suicidal behavior is common in patients with psychiatric disorders within the spectrum of depressive features, in this study we measured soluble interleukin-2 receptor concentrations in plasma (sIL-2R) and investigated temperament and…

Rothenhausler, Hans-Bernd; Stepan, Alexandra; Kapfhammer, Hans-Peter

2006-01-01

83

Nisin-Controlled Extracellular Production of Interleukin-2 in Lactococcus lactis Strains, without the Requirement for a Signal Peptide Sequence?  

PubMed Central

Secretion of the cytokine interleukin-2 (IL-2) was investigated in Lactococcus lactis using the secretory machinery of the bacteriocin lactococcin A. Surprisingly, the lcnCD transport genes were not essential for mouse IL-2 secretion. Furthermore, expression of a mature mouse IL-2 gene resulted in interleukin secretion without the requirement for a leader sequence. PMID:17905884

Fernández, Antonio; Rodríguez, Juan M.; Bongaerts, Roy J.; Gasson, Michael J.; Horn, Nikki

2007-01-01

84

Effects of Interleukin 2 Receptor Blockers on Patient and Graft Survival in Renal-Transplanted Children  

PubMed Central

Background: Monoclonal antibodies block interleukin-2 receptors on alloantigen-reactive T-Lymphocytes and induce selective immunosuppression. It is postulated that induction therapy with these agents in pediatric transplantation may decrease acute rejection and improve graft survival with no significant side effect or increase in the incidence of viral infections. Objectives: The aim of this study was to examine the effects of interleukin 2 receptor blockers on patient and graft survival in renal-transplanted children. Patients and Methods: One hundred and eighty six children aged 7-13 years who received renal transplantation in university-affiliated hospital between 2003 and 2012 were enrolled in the study. All patients received prednisolone, cyclosporine and mycophenolate mofetil or azathioprine as basic immunosuppressive therapy. Patients were divided into two groups according to receiving induction therapy with IL2-receptor blockers. We investigated for acute rejection episodes, Cytomegalovirus (CMV) and BK virus infection and one and three year’s survival of the patients and the grafts Results: From 186 renal-transplanted children included in this study, 36 patients were in treated group (group 1) and 150 patients in control group (group 2). The mean age of the patients was 10.4 ± 2 years and 55.6% were males. In first six months of transplantation, eight patients in group one had one episode of acute rejection and no one had two episodes. Early acute rejection rate was 8.36 (22%). In the control group, 37 patients had one episode and three patients had two episodes of acute rejection (rejection rate 28.6%). Therefore, early acute rejection rates were lower in group one. Late acute rejection rates did not show any difference in group 1 and group 2 (27.7% vs. 27.3% respectively). There was lower prevalence of steroid-resistance rejection in group 1 patients (5.5%) compared with 6.6% in group 2, but it did not reach statistical significance. None of the patients in IL2-R blocker group died at one year follow-up (patient survival 100%). However, in control group, four (2.6%) patients died toward the end of first year (patient survival 97.4%). When patients in group 1 and group 2 were age and sex matched with equal number the difference was significant (P < 0.05). Conclusions: Induction therapy with IL2-R blockers reduced the rate of early acute rejection, but had no effect on late acute rejections. Patient and graft survival were better in treated group, but did not reach statistical significance. A longer period of follow-up may be required to discern a clear advantage for induction therapy with these agents. PMID:25695021

Sharifian, Mostafa; Arad, Banafsheh; Simfroosh, Naser; Basiri, Abbas; Otukesh, Hassan; Esfandiar, Nasrin

2014-01-01

85

Interferon-gamma and interleukin-2 stimulate production of a soluble factor by L1210 and P815 tumor targets to promote macrophage activation.  

PubMed

Previously it was established that L1210 mouse leukemia and a variety of other tumor cell types produced a soluble factor(s), designated tumor-derived recognition factor (TDRF), which synergized with interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) or lipopolysaccharide (LPS) to promote increased tumor necrosis factor-alpha (TNF-alpha) and nitric oxide synthase (NOS) mRNA synthesis by murine macrophages (M phi). Other work revealed that pretreatment of L1210 tumor targets with IFN-gamma rendered them more susceptible to NO-mediated killing by LPS-activated M phi. Now we have combined these observations to determine if pretreatment of L1210 or P815 tumor targets with IFN-gamma and/or IL-2 would augment production of TDRF to enhance M phi activation. Results confirmed that pretreatment of either L1210 or P815 targets with 200 u/ml of IFN-gamma or 1,000 u/ml of IL-2 significantly increased their susceptibility to M phi-mediated cytotoxicity owing to increased NO production. Similar pretreatment of L1210 targets with suboptimal concentrations of IFN-gamma and IL-2 in combination resulted in additive rather than synergistic augmentation of NO-mediated cytotoxicity by cytotoxic M phi. Pretreatment of L1210 targets with IFN-gamma or IL-2 alone or in combination increased the production of TDRF above constitutive levels as demonstrated by increased production of NO and induction of NOS mRNA expression by cytotoxic M phi. Thus IFN-gamma and/or IL-2 promoted increased TDRF production by tumor targets which in turn promoted M phi generation of tumor cytotoxic NO. It appears that M phi activating cytokines have a dual role in acting on certain tumor targets to augment the process of M phi activation through the increased elicitation of immunopotentiating tumor-derived soluble factor(s). PMID:8852603

Jiang, H; Robinson, W E; Leu, R W

1996-01-01

86

The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system  

SciTech Connect

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-?, tumor necrosis factor-?, interleukin (IL)-1?, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ? and ? subunits of IL-2 receptor (IL-2R), while the mRNA levels of the ? subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2R? subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ? Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ? Er-1 increases the T-cell production of specific cytokines. ? Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ? The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

Cervia, Davide, E-mail: d.cervia@unitus.it [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy) [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Catalani, Elisabetta; Belardinelli, Maria Cristina [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Perrotta, Cristiana [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy)] [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Picchietti, Simona [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Alimenti, Claudio [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)] [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy); Casini, Giovanni; Fausto, Anna Maria [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Vallesi, Adriana [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)] [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)

2013-02-01

87

Interleukin-2 Expression in Lupoid and Usual Types of Old World Cutaneous Leishmaniasis  

PubMed Central

Background: Interleukin (IL)-2 plays a central role in T cell-dependent immune responses. Objectives: We conducted this study to determine and compare IL-2 expression in lupoid and usual types of Old World Cutaneous Leishmaniasis (OWCL), using immunohistochemistry. Patients and Methods: Thirteen paraffin-embedded specimens of lupoid and 12 specimens of usual types of OWCL were used. A mouse monoclonal anti IL-2 antibody was used for staining by the envision technique. Results: There were strongly stained discrete foci of staining through inflammatory infiltrates of dermis and also in basal layers of epidermis and adnexal structures, with a distinctive pattern of hot spot activity foci (mean of 9.31 ± 6.4 versus 8.17 ± 6.9 foci per HPF for lupoid and usual types, respectively). The expression of IL-2 had no correlation with the pattern of granulomatous inflammation (tuberculoid, sarcoidal or mixed suppurative). Conclusions: Interleukin-2 takes part in the immunological response of the granulomatous reaction of OWCL and is not statistically different between lupoid and usual types (P = 0.674).

Mashayekhi Goyonlo, Vahid; Elnour, Hesameldin; Nordlind, Klas

2014-01-01

88

Ectodomain Shedding of Interleukin-2 Receptor ? and Generation of an Intracellular Functional Fragment*  

PubMed Central

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-R?, -?, and -?c). IL-2R? is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2R? is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37?ic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2R? decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37?ic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2R? mimicking 37?ic increases their proliferation. These data indicate that IL-2R? is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation. PMID:20495002

de Oca B., Pavel Montes; Malardé, Valerie; Proust, Richard; Dautry-Varsat, Alice; Gesbert, Franck

2010-01-01

89

Role of Interleukin-2 in Uremic Pruritus Among Attendants of AL-Zahraa Hospital Dialysis Unit  

PubMed Central

Background: Uremic pruritus (UP) is a very distressing symptom and remains one of the most frustrating and potentially disabling symptoms in patients with end-stage renal disease (ESRD). Its etiopathogenesis remains unclear and complex. The aim of this study was to investigate the possible role of interleukin-2 (IL-2) in UP, and correlate its level with the severity of itching in ESRD patients. Patients and Methods: This study was carried out on 60 patients on maintenance hemodialysis (HD), 30 patients with UP and 30 patients without UP, and 30 apparently healthy age- and sex-matched subjects as controls. Itch intensity was scored as mild, moderate, and severe using five-dimensional itch scale. Some relevant clinical parameters (age, sex, xerosis, presence of neuropathy, duration of dialysis, complete medical history, and history of pruritic skin diseases) and laboratory findings including creatinine, urea, calcium, phosphorus, parathyroid hormone, and serum levels of IL-2 were evaluated. Results: In our study, we found a statistically significant difference in IL-2 level between patients and controls. However, there was no statistically significant difference in IL-2 levels between cases with pruritus and cases without pruritus. Also, there was a statistically significant relation between IL-2 level and duration of the disease. Conclusion: Further studies are needed to understand the contribution of IL-2 and possibly other cytokines in the pathogenesis of this distressing symptom in ESRD. PMID:25814728

Azim, Amira Adel Abdel; Farag, Asmaa Saied; El-Maleek Hassan, Doaa Abd; Abdu, Safaa Mahmoud Ismail; Lashin, Somaya Mohamed Abo-Elfetouh; Abdelaziz, Nahla Mohamed

2015-01-01

90

[Expression of goose interleukin-2 gene in Escherichia coli and isolation of its soluble monomer].  

PubMed

Recombinant expression plasmid of pET-28a (+)-goIL-2 was constructed by inserting the goose IL-2 gene without the signal peptide sequence into the prokaryotic expression vector pET-28a (+), and transformed into the bacterial competent E. coli BL21 (DE3) cells for expression. After IPTG induction, an expected protein band with molecular weight of 15.0 kD was observed on SDS-PAGE gel, recognized by monoclonal antibody against goose IL-2 in western-blotting assay. In the pET-28a (+) expression system, much of the recombinant goose IL-2 (rgoIL-2) was found in inclusion bodies with a portion of soluble protein. The monomer and multimers of soluble goose interleukin 2 proteins were observed in native electrophoresis. The rgoIL-2 proteins were purified by Ni-NTA column under a native condition. The rgoIL-2 soluble protein monomer was isolated by a quick protein isolation and purification system of AKTA FPLC and identified by native PAGE. Bioactivity analysis showed that the rgoIL-2 monomer stimulated the proliferation of goose lymphocytes in vitro. This will establish a basis for further study about the biological function and clinical application of goose IL-2. PMID:18464597

Qi, Jing; Chen, Jigang; Wang, Jinyong; Fang, Jie; Wu, Jiajun; Zhou, Jiyong

2008-02-01

91

Recombinant human interleukin-2-induced mitogenic proliferation of in vitro unstimulated bovine intestinal lymphocytes.  

PubMed Central

Recombinant human interleukin-2 (rHIL-2) in the absence or presence of additional stimuli, was able to induce and support the proliferation of lymphocytes isolated from the intra-epithelium, lamina propria and Peyer's patches of the small intestine of normal adult cows. Although dose-dependent effects of rHIL-2 were observed with all three cell populations, concentrations as low as 2.5 U/mL were able to induce DNA synthesis as measured by tritiated thymidine incorporation. Furthermore, rHIL-2 as low as 5.0 U/mL was shown to significantly enhance lymphocyte proliferation in response to mitogenic stimulation. These proliferative responses to rHIL-2 were detected within two days of culture and peaked after five days. Although the extent of the blastogenic response was variable in individual animals, the general pattern of time-course and dose-response to rHIL-2 was similar in all animals tested. The response of all three leukocyte populations to rHIL-2 was dependent on the presence of adherent accessory cells and/or 2-mercaptoethanol. Both nylon wool nonadherent (T cells, null cells) and adherent cells (B cells) were shown to be responsive to rHIL-2. These studies demonstrate that bovine lymphocytes isolated from different anatomical locations of the small intestine are capable of proliferation in response to xenogenic IL-2 without in vitro preactivation signals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2783657

Nagi, A M; Babiuk, L A

1989-01-01

92

Interleukin-2-inducible T cell kinase regulates mast cell degranulation and acute allergic responses.  

PubMed

Bruton's tyrosine kinase (Btk) is thought to positively regulate mast cell activation, implying a role in allergic responses. We have compared acute and late phase allergic airway reactions in mice lacking either Btk or interleukin-2-inducible T cell kinase (Itk), another Tec kinase expressed in mast cells. Btk(-/-) mice showed minor protection against allergic symptoms when challenged with allergen via the airways. In sharp contrast, both acute and late phase inflammatory allergic responses were markedly reduced in Itk(-/-) mice. Notably, airway mast cell degranulation in Itk(-/-) mice was severely impaired, despite wild-type levels of allergen-specific IgE and IgG1. The degranulation defect was confirmed in DNP-conjugated human serum albumin-challenged mice passively sensitized with anti-DNP IgE antibodies, and was also observed after direct G-protein stimulation with the mast cell secretagogue c48/80. Moreover, late phase inflammatory changes, including eosinophilia, lymphocyte infiltration, and Th2 cytokine production in the lungs, was eliminated in Itk(-/-) mice. Collectively, our data suggest a critical role of Itk in airway mast cell degranulation in vivo that together with an impaired T cell response prevents the development of both acute and late phase inflammatory allergic reactions. PMID:15778496

Forssell, Johan; Sideras, Pascalis; Eriksson, Christina; Malm-Erjefält, Monika; Rydell-Törmänen, Kristina; Ericsson, Per-Olof; Erjefält, Jonas S

2005-06-01

93

The selective ablation of interleukin 2-producing cells isolated from transgenic mice  

PubMed Central

To better understand the requirement for interleukin 2 (IL-2) in specific immune responses, we have established the use of cell ablation to selectively eliminate T cells that produce IL-2. To accomplish this we have generated transgenic mice that express the herpes simplex virus 1-thymidine kinase (HSV-TK) gene under the transcriptional control of the murine IL-2 promoter that renders IL-2-producing cells sensitive to the cytotoxic effects of the antiviral drug ganciclovir (GANC). HSV-TK activity was specifically expressed in activated T cells from transgenic mice. When CD4 T cells from transgenic mice were stimulated with the superantigen staphylococcal enterotoxin A (SEA) in the presence of GANC, proliferation and IL-2 production were almost completely inhibited and the activated CD4+V beta 3+ T cell population, eliminated. Proliferation was not restored by adding IL-2, showing that most proliferating cells are not bystander cells. In contrast, the proliferative response to concanavalin A (Con A) was only partially inhibited by treatment of CD4 T cells with GANC, although the efficiency of eliminating IL-2-producing cells was shown to be comparable with that achieved using SEA. This suggests that a portion of the proliferative response to Con A occurs via an alternative pathway not requiring IL-2 synthesis and release. PMID:8386745

1993-01-01

94

The development of new immunotherapies for the treatment of cancer using interleukin-2. A review.  

PubMed Central

Recent increases in knowledge of cellular immunology, combined with developments in biotechnology, have provided new opportunities for the development of immunotherapies for the treatment of cancer in humans. One approach to therapy is that of adoptive immunotherapy, that is, the transfer to the tumor bearing host of lymphoid cells with antitumor reactivity that can mediate antitumor responses. Several lymphocyte subpopulations have now been identified that may be suitable for use in adoptive immunotherapy. Resting lymphocytes incubated in interleukin-2 (IL-2) give rise to lymphokine activated killer (LAK) cells that can lyse malignant cells, but not normal cells. Clinical studies in patients with advanced cancer have revealed that treatment with high dose IL-2 alone or in combination with LAK cells can mediate the complete or partial regression of cancer in selected patients. Other approaches are currently undergoing investigation, including the adoptive transfer of tumor infiltrating lymphocytes, which, in animal models, have antitumor reactivity 50-100 times more potent than do LAK cells. Other new approaches to immunotherapy include the use of combination of lymphokines, such as the use of tumor necrosis factor or alpha interferon in conjunction with IL-2. The availability of recombinant lymphokines that provide large amounts of biologically active materials can hopefully lead to the development of effective new therapies for cancer in humans. Images Fig. 4. Fig. 5. Figs. 6A and B. Fig. 7. Figs. 8A and B. Fig. 9. Fig. 10. Fig. 11. Figs. 12A and B. Figs. 13. Fig. 14. Fig. 15. PMID:3041925

Rosenberg, S A

1988-01-01

95

Soluble interleukin-2 receptor, intercellular adhesion molecule-1 and interleukin-10 serum levels in patients withelanoma  

PubMed Central

Serum soluble interleukin-2 receptor (sIL-2R), intercellular adhesion molecule-1 (sICAM-1) and interleukin-10 (IL-10) have each been reported as useful markers for melanoma progression. To evaluate the clinical relevance of these three markers, we simultaneously analysed their serum levels in patients with melanoma. A longitudinal study with a 3-year follow-up was performed and different stages of the disease were considered. Mean values of sIL-2R were significantly higher than in normal controls in all stages and correlated with the disease progression. The prognosis of patients with levels > 529 U/ml of sIL-2R was significantly poorer than in patients with sIL-2R levels < 529 U/ml. Levels of sICAM-1 were also elevated in melanoma patients, specially at the time of the metastatic disease. Serum IL-10 levels were more frequently detectable in the patients that developed metastasis during follow-up, and the prognosis of patients with detectable IL-10 levels was significantly poorer than in those patients with IL-10 undetected levels. Statistical analysis based on Logistic and Cox regression models showed that only sex, stage and sIL-2R value are factors significantly associated with metastatic progression. Moreover, high levels of sIL-2R could be a risk factor for malignant progression in melanoma. © 2000 Cancer Research Campaign PMID:10970683

Boyano, M D; Garcia-Vázquez, M D; López-Michelena, T; Gardeazabal, J; Bilbao, J; Cañavate, M L; Galdeano, A García De; Izu, R; Díaz-Ramón, L; Raton, J A; Díaz-Pérez, J L

2000-01-01

96

Green tea polyphenol extract attenuates inflammation in interleukin-2-deficient mice, a model of autoimmunity.  

PubMed

Green tea polyphenols (GrTP) have been previously shown to decrease endotoxin-induced tumor necrosis factor-alpha production and lethality in mice. Our present studies demonstrate that GrTP inhibit inflammatory responses and may be useful in treating chronic inflammatory states, such as inflammatory bowel disease. In this preliminary study, we examined whether GrTP decrease disease activity in interleukin-2-deficient (IL-2(-/-) mice. Eight-week old IL-2(-/-) C57BL/6J mice who were fed nonpurified diet were randomly assigned to receive water with GrTP (5 g/L) or water alone (control) for up to 6 wk. After 1 wk, explant colon and lamina propria lymphocyte (LPL) cultures were established from a subgroup of mice and supernatants collected. Culture supernatants from GrTP-treated mice showed decreased spontaneous interferon-gamma and tumor necrosis factor-alpha secretion compared with that of controls. At 6 wk, the GrTP group had less severe colitis as demonstrated by lower histologic scores and wet colon weights. This was associated with lower plasma levels of serum amyloid A, increased weight gain and improved hematocrits. These results show that GrTP attenuated inflammation in IL-2(-/-) mice and suggest a role for GrTP in treating chronic inflammatory diseases such as inflammatory bowel disease. PMID:11435526

Varilek, G W; Yang, F; Lee, E Y; deVilliers, W J; Zhong, J; Oz, H S; Westberry, K F; McClain, C J

2001-07-01

97

Activation of endothelium by immunotherapy with interleukin-2 in patients with malignant disorders.  

PubMed

Treatment with intravenous recombinant human interleukin-2 (rh IL-2) is frequently accompanied by the capillary leak syndrome and disturbances of the coagulation system. Although the exact mechanisms are still not fully understood, the involvement of the endothelium is proven. This investigation aimed to elucidate more precisely the role of the endothelium in the generation of IL-2-based side-effects. In nine tumour patients receiving intravenous rh IL-2, parameters characterizing endothelial cell activation as well as activation of the coagulation system were evaluated. A significant increase of the circulating endothelial leucocyte adhesion molecule-1 (cELAM-1) and the vasoconstrictor peptide endothelin-1 (ET-1) was observed (P<0.05), indicating activation of endothelial cells. The simultaneous increase of tissue-plasminogen activator and plasminogen activator inhibitor type-1 during therapy (P<0.05) corroborated this observation. A decrease in platelet count parallelled by an increase of fibrin degradation products, the prolongation of partial thromboplastin time, and the decrease of fibrinogen (P<0.05) suggested the development of disseminated intravascular coagulation (DIC), induced by activated endothelium and intensified by transient hepatic failure. We concluded that activation of the endothelium mediated by IL-2 was accompanied by a loss of endothelial integrity and capillary leak. The activated endothelium can trigger DIC via activation of the coagulation cascade. The increased ET-1 might act as an endogenous counter-regulator of the disadvantageous haemodynamic side-effects induced by IL-2. PMID:10554800

Locker, G J; Kapiotis, S; Veitl, M; Mader, R M; Stoiser, B; Kofler, J; Sieder, A E; Rainer, H; Steger, G G; Mannhalter, C; Wagner, O F

1999-06-01

98

Interleukin 2 in the pathogenesis and therapy of type 1 diabetes.  

PubMed

Regulatory T cells (Tregs) play a major role in controlling effector T cells (Teffs) responding to self-antigens, which cause autoimmune diseases. An improper Treg/Teff balance contributes to most autoimmune diseases, including type 1 diabetes (T1D). To restore a proper balance, blocking Teffs with immunosuppressants has been the only option, which was partly effective and too toxic. It now appears that expanding/activating Tregs with low-dose interleukin-2 (IL-2) could provide immunoregulation without immunosuppression. This is particularly interesting in T1D as Tregs from T1D patients are reported as dysfunctional and a relative deficiency in IL-2 production and/or IL-2-mediated signaling could contribute to this phenotype. A clinical study of low-dose IL-2 showed a very good safety profile and good Treg expansion/activation in T1D patients. This opens the way for efficacy trials to test low-dose IL-2 in prevention and treatment of T1D and to establish in which condition restoration of a proper Treg/Teff balance would be beneficial in the field of autoimmune and inflammatory diseases. PMID:25344788

Rosenzwajg, Michelle; Churlaud, Guillaume; Hartemann, Agnès; Klatzmann, David

2014-12-01

99

A novel and simple type of liposome carrier for recombinant interleukin-2.  

PubMed

The strong interaction between recombinant interleukin-2 (IL-2) and liposome was characterized and its possible application to drug-delivery control considered. The liposomes were prepared with egg phosphatidylcholine, distearoyl-phosphatidylglycerol (DSPG), dipalmitoyl-phosphatidylcholine, dipalmitoyl-phosphatidylglycerol or distearoyl-phosphatidylcholine (DSPC). Small and hydrophobic liposomes were selected, which were composed of saturated and long-fatty-acid-chain phospholipids. When the composition and the mixture ratio of IL-2 and the liposomewere optimized, morethan 95% ofthe lyophilized IL-2 (Imunace, 350000 JRU) was adsorbed consistently onto the DSPC-DSPG liposome (molar ratio, 10:1; 25 micromol mL(-1); 30 nm in size). Merely mixing IL-2 lyophilized with liposome suspension is convenient pharmaceutically. After intravenous administration to mice, liposomal IL-2 was eliminated half as slowly from the systemic circulation as free IL-2, with more than 13 and 18 times more IL-2 being delivered to the liver and spleen, respectively. After subcutaneous administration of liposomal IL-2 to mice, the mean residence time of IL-2 in the systemic circulation was 8 times that of free IL-2. These results show that IL-2 consistently adsorbs onto the surface of liposomes after optimization of its composition and mixing ratio. Intravenous and subcutaneous administration to mice demonstrates the gradual release of IL-2. Further trials are warranted using these liposomes. PMID:11291744

Kanaoka, E; Takahashi, K; Yoshikawa, T; Jizomoto, H; Nishihara, Y; Hirano, K

2001-03-01

100

Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions.  

PubMed Central

Known transcription factor-DNA interactions in the minimal enhancer of the murine interleukin-2 gene (IL-2) do not easily explain the T cell specificity of IL-2 regulation. To seek additional determinants of cell type specificity, in vivo methodologies were employed to examine chromatin structure 5' and 3' of the 300 bp IL-2 proximal promoter/enhancer region. Restriction enzyme accessibility revealed that until stimulation the IL-2 proximal promoter/enhancer exists in a closed conformation in resting T and non-T cells alike. Within this promoter region, DMS and DNase I genomic footprinting also showed no tissue-specific differences prior to stimulation. However, DNase I footprinting of the distal -600 to -300 bp region revealed multiple tissue-specific and stimulation-independent DNase I hypersensitive sites. Gel shift assays detected T cell-specific complexes binding within this region, which include TCF/LEF or HMG family and probable Oct family components. Upon stimulation, new DNase I hypersensitive sites appeared in both the proximal and distal enhancer regions, implying that there may be a functional interaction between these two domains. These studies indicate that a region outside the established IL-2 minimal enhancer may serve as a stable nucleation site for tissue-specific factors and as a potential initiation site for activation-dependent chromatin remodeling. PMID:9611237

Ward, S B; Hernandez-Hoyos, G; Chen, F; Waterman, M; Reeves, R; Rothenberg, E V

1998-01-01

101

Serum levels of soluble interleukin-2 receptor alpha (sIL2R?) as a predictor of outcome in brucellosis  

Microsoft Academic Search

Objectives and methodsBrucellosis is characterized by chronicity and relapses despite efficacious treatment. Cytokines and especially the Th1\\/Th2 balance may be involved in the susceptibility or resistance to the Brucella species. In order to identify predictors of treatment outcome, we measured the pre and posttreatment levels of serum interleukin-2 (IL-2) and soluble IL-2 receptor alpha (sIL-2R?) in 20 children with brucellosis.

Alexandros C. Makis; Emmanouil Galanakis; Eleftheria C. Hatzimichael; Zoe L. Papadopoulou; Antigone Siamopoulou; Konstantinos L. Bourantas

2005-01-01

102

Results of Immunochemotherapy with Interleukin2, Interferon-?2 and 5Fluorouracil in the Treatment of Metastatic Renal Cell Cancer  

Microsoft Academic Search

Objective: In patients with advanced metastatic renal cell carcinoma (RCC) seen at a single institution, the toxicity and long-term clinical effects of a combination therapy with recombinant interleukin-2 (rIL-2), recombinant interferon-?2 (rIFN-?2) and 5-fluorouracil (5-FU) were evaluated. Method: From August 1992 through August 1997, 47 consecutive patients (38 men) with metastatic RCC were treated using rIL-2 and rIFN-?2 subcutaneously in

Dirk Samland; Frank Steinbach; Frank Reiher; Uwe Schmidt; Achim Gruss; Ernst P. Allhoff

1999-01-01

103

Kappa B--Specific DNA Binding Proteins: Role in the Regulation of Human Interleukin2 Gene Expression  

Microsoft Academic Search

Transcriptional activation of the human interleukin-2 (IL-2) gene, like induction of the IL-2 receptor alpha (IL-2Ralpha ) gene and the type 1 human immunodeficiency virus (HIV-1), is shown to be modulated by a kappa B-like enhancer element. Mutation of a kappa B core sequence identified in the IL-2 promoter (-206 to -195) partially inhibits both mitogen- and HTLV-I Tax-mediated activation

Beatrice Hoyos; Dean W. Ballard; Ernst Bohnlein; Miriam Siekevitz; Warner C. Greene

1989-01-01

104

Phase II study of interleukin-2 and 13- cis -retinoic acid as maintenance therapy in metastatic colorectal cancer  

Microsoft Academic Search

Purpose  We have previously shown that low-dose interleukin-2 (IL-2) and 13-cis-retinoic acid (13-cis-RA) improved lymphocyte and natural killer (NK) cell count of patients with advanced tumors showing a clinical benefit from\\u000a chemotherapy. The primary endpoint of this study was to ask whether IL-2 and 13-cis-RA improved (-0%) lymphocyte and NK cell count in patients with metastatic colorectal cancer (MCRC) that had

Francesco Recchia; Gaetano Saggio; Alisia Cesta; Giampiero Candeloro; Anna Di Blasio; Giovanna Amiconi; Marco Lombardo; Antonio Nuzzo; Angelo Lalli; Edoardo Alesse; Stefano Necozione; Silvio Rea

2007-01-01

105

The cognitive effects of recombinant interleukin-2 (rIL-2) therapy: a controlled clinical trial using computerised assessments  

Microsoft Academic Search

It has been suggested that patients undergoing treatment with recombinant interleukin-2 (rIL-2) may develop cognitive impairment. To evaluate these effects, 17 patients with advanced colorectal cancer took part in a randomised, parallel group study of rIL-2 with chemotherapy (5-fluorouracil and leucovorin) and chemotherapy alone. Assessments were carried out daily whilst patients were in hospital and regularly between cycles of treatment

L. G. Walker; K. P. Wesnes; S. D. Keys; M. B. Walker; J. Lolley; O. Eremin

1996-01-01

106

Two mutational hotspots in the interleukin-2 receptor γ chain gene causing human X-linked severe combined immunodeficiency  

Microsoft Academic Search

Human severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immunity, is most commonly caused by mutations in the X-linked gene for interleukin-2 (IL-2) receptor γ chain (IL2RG). For mutational analysis of IL2RG in males with SCID, SSCP screening was followed by DNA sequencing. Of 40 IL2RG mutations found in unrelated SCID patients, 6 were point mutations

A. E. Pepper; J. M. Puck; R. H. Buckley

1995-01-01

107

Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism  

PubMed Central

Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-? and TNF-? production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides. PMID:23840803

Gründemann, Carsten; Thell, Kathrin; Lengen, Karin; Garcia-Käufer, Manuel; Huang, Yen-Hua; Huber, Roman; Craik, David J.; Schabbauer, Gernot; Gruber, Christian W.

2013-01-01

108

Suppression of multiple bioactivities of interleukin-1 and interleukin-2 production by U937 conditioned medium  

SciTech Connect

The human macrophage-like cell line U937 spontaneously produces a factor which blocks interleukin-1 (IL-1) activity for mouse thymocytes but not mitosis of T-lymphoblastoid cells. The authors examined the effects of U937 conditioned medium (CM) on other IL-1 activities and on interleukin-2 (IL-2) production. U937 was cultured at 5 x 10/sup 6//ml in RPMI-1640 at 37/sup 0/C for 5 days. The resulting CM inhibited the mitogenic response of C3H/HeJ mouse thymocytes to an IL-1 standard, with an inhibitory of activity of 6.64 U/ml (1 U = reciprocal dilution producing 50% inhibition of maximal response). Similarly, CM inhibited (10.12 U/ml) the fibroblast stimulation promoter activity of IL-1. The effect of CM on IL-2 production was assessed in a direct assay in which IL-2 production by ..gamma..-irradiated (12,000 rads) MLA-144 lymphosarcoma cells was assayed as /sup 3/H-thymidine incorporation in CTLL-20 cells. The suppressive activity of CM was 4.95 U/ml; CM did not interfere with the response of CTLL-20 to IL-2. These studies establish that U937 produces factors with multiple, related biological activities; U937 CM blocks IL-2 dependent (thymocyte mitogenesis) and IL-2 independent (fibroblast proliferation) IL-1 activities and interferes with production of, but not response to, IL-2. U937 is an excellent model to study growth inhibitory properties of mononuclear phagocytes.

Wiblin, R.T.; Edmonds, K.; Ellner, J.J.

1986-03-01

109

Systemic Administration of Interleukin 2 Enhances the Therapeutic Efficacy of Dendritic Cell-Based Tumor Vaccines  

NASA Astrophysics Data System (ADS)

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of non-toxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-? production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

Shimizu, K.; Fields, R. C.; Giedlin, M.; Mule, J. J.

1999-03-01

110

Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes.  

PubMed

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining "suicide" and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6-23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient "bystander" effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK+ cells, some TK- cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man. PMID:10414462

Soler, M N; Milhaud, G; Lekmine, F; Treilhou-Lahille, F; Klatzmann, D; Lausson, S

1999-01-01

111

Abnormal expression of interleukin-2 receptor (Tac antigen) in adult T-cell leukemia.  

PubMed

Human T-cell leukemia/lymphoma virus type I(HTLV-I) infection appears to be closely associated with the leukemogenesis of adult T-cell leukemia (ATL), although its mechanism remains unclear. Since our previous report that leukemic cells from patients with ATL expressed Tac antigen (Ag) (interleukin-2 (IL-2) receptor) on their cell surface, we have been studying IL-2 receptors on ATL leukemic cells to see whether they are different from normal IL-2 receptors and whether they play a role in the neoplastic growth of ATL cells. Peripheral blood leukemic cells from 35 patients with ATL expressed IL-2 receptors which were detected by anti-Tac monoclonal antibody when examined immediately after the separation of cells or after culture for 24 or 48 hr. The number of anti-Tac binding sites ranged from 4,300 to 11,400 in fresh cells and from 6,100 to 96,000/cell in short term cultured leukemic cells, whereas phytohemagglutinin-P(PHA-P)-stimulated normal T cells exhibited 7,000 to 35,000 anti-Tac binding sites/cell. HTLV-I-infected cell lines such as MT-1 and HUT-102 expressed a markedly enhanced number of Tac Ag molecules. Leukemic cells from 15 ATL patients showed no or very poor proliferative response to various concentrations of IL-2, although they expressed Tac Ag. Radiolabeled IL-2 binding experiments revealed that ATL leukemic cells could bind IL-2 although the number of IL-2 binding sites was much less than that of anti-Tac binding sites. IL-2 receptors on ATL cells, unlike normal activated T cells, were not modulated (down-regulated) by anti-Tac antibody.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6100643

Uchiyama, T; Wano, Y; Tsudo, M; Umadome, H; Hori, T; Tamori, S; Uchino, H; Yodoi, J; Maeda, M; Kobayashi, N

1984-01-01

112

Adult T leukemia cells produce a lymphokine that augments interleukin 2 receptor expression.  

PubMed

Human T-cell leukemia virus (HTLV)-infected cell lines derived from adult T-cell leukemia (ATL) express constitutively the receptor for Interleukin-2 (IL-2-R) and the associated antigen (Tac antigen). In contrast, the same antigen is transiently expressed by normal T-cells only after immune stimulation. Recently, it was reported that the constitutively expressed Tac antigen on ATL cells and cell lines was not down-regulated or modulated by anti-Tac antibody. Since the antigen was modulated on normal mitogen- or alloantigen-stimulated T-cells, we postulated that the regulation of IL-2-R may be abnormal on ATL cells; the synthesis of IL-2-R is continuously stimulated in these cells. A unique HTLV/ATLV(-) cell line (YT) derived from a child with acute lymphoblastic leukemia was found to express low levels of Tac antigen that could be enhanced by various stimuli, including conditioned medium (CM) derived from normal lymphocytes, but not by lectins (PHA, Con A). Of particular interest, the exposure of YT cells to CM from ATL cell lines with helper phenotype revealed the presence of factor(s) (ATL-derived factor, ADF) that augmented the synthesis and expression of IL-2-R/Tac antigen on YT cells and promoted YT cell growth. CM from HTLV(-) leukemia cell lines lacked both IL-2-R augmenting activity and a growth promoting activity. Immunoaffinity-purified IL-2 and recombinant gamma interferon also lacked IL-2-R augmenting activity. Moreover, the physicochemical analysis with Fast protein liquid chromatography (FPLC) revealed that ADF was quite different in pI point from the IL-2-R augmenting activity in CM from normal lymphocytes. These results suggested that ADF is a unique product of HTLV(+) cells. The possible relationship between ADF production, HTLV infection, and the abnormal expression of IL-2-R is suggested, and these abnormalities may be advantageous for the leukemogenesis and abnormal growth of ATL. PMID:2978223

Teshigawara, K; Maeda, M; Nishino, K; Nikaido, T; Uchiyama, T; Tsudo, M; Wano, Y; Yodoi, J

1985-01-01

113

Transcriptional regulation by retinoic acid of interleukin-2 alpha receptors in human B cells.  

PubMed Central

In this study, we demonstrated that retinoic acid (RA) up-regulated interleukin-2 receptor-alpha (IL-2R alpha) expression on two human B-cell lines, IE8.6 and SKW6.4. Deleted forms of the human IL-2R alpha promoter linked to the bacterial chloramphenicol acetyltransferase reporter gene were transfected into IE8.6 cells in order to define RA-responsive regulatory domains. Experiments using the -1.6 kb construct, which contains all known regulatory regions in the IL-2R alpha promoter, indicated that RA could induce IL-2R alpha promoter activity. The basal activity of the -471 construct was initially low, but was markedly enhanced by the addition of RA. Deletion of promoter sequences between -471 and -317 resulted in a significant augmentation of basal promoter activity and abolished promoter induction by RA. This finding revealed a requirement for sequences 5' of base -317 for RA-induced promoter activation, raising the possibility of the presence of both a RA response element and a negative regulatory element (NRE) upstream of base -317. Transfection studies with internal deletion mutants with the putative NRE removed resulted in increases in basal promoter activity and unresponsiveness to RA similar to the -317 construct. In contrast, an internal deletion mutant with the NRE intact had low basal activity and was inducible by RA similar to the -471 construct. Taken together, our results suggested that RA-induced activation of the IL-2R alpha promoter was through changes in the function of a NRE present between bases -400 and -368. This 31-base pair element may interact with an adjacent RA-responsive regulatory site as well as being responsible for down-regulation of basal IL-2R alpha expression under certain conditions. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8157276

Bhatti, L; Sidell, N

1994-01-01

114

Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.  

PubMed Central

Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction. Images PMID:1976256

Burton, J; Goldman, C K; Rao, P; Moos, M; Waldmann, T A

1990-01-01

115

Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes.  

PubMed Central

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R. PMID:2004488

Tomizawa, K; Ishizaka, A; Kojima, K; Nakanishi, M; Sakiyama, Y; Matsumoto, S

1991-01-01

116

Bronchoalveolar lavage analysis, gallium-67 lung scanning and soluble interleukin-2 receptor levels in asbestos exposure  

SciTech Connect

This study examined different markers of lung immunologic and inflammatory responses to previous asbestos exposure. We performed bronchoalveolar lavage (BAL) and gallium-67 (/sup 67/Ga) lung scans and measured serum and BAL soluble interleukin-2 receptor (IL-2R) and angiotensin-converting enzyme (SACE) levels in 32 subjects with a history of significant asbestos exposure, 14 without (EXP) and 18 with (ASB) radiographic evidence of asbestosis. BAL analysis revealed increases in neutrophils in both ASB and EXP when compared to controls (P less than 0.01), which persisted after adjustment for smoking category. Although significant abnormalities of macrophage and total lymphocyte profiles were not found in the study population, lymphocyte subpopulation analysis revealed elevation of BAL T4/T8 ratios in the entire study group (ASB + EXP) when compared to controls (P less than 0.05), independent of smoking category. /sup 67/Ga lung scan activity was increased in 56% of ASB and in 36% of EXP: no correlations between positive scans and different radiological and functional parameters could be found. There was no significant elevation of mean SACE, serum, or BAL IL-2R levels in any of the study categories. These data suggest that asbestos exposure may be associated with parenchymal inflammation, even in the absence of clinical criteria for asbestosis. Abnormalities of gallium uptake and of BAL analysis reflect the clinically inapparent inflammation. The increased BAL T4/T8 ratios observed suggest that abnormal local pulmonary immunoregulation may play a role in the pathogenesis of asbestos-related lung diseases.

Delclos, G.L.; Flitcraft, D.G.; Brousseau, K.P.; Windsor, N.T.; Nelson, D.L.; Wilson, R.K.; Lawrence, E.C.

1989-04-01

117

Role of the interleukin 2 receptor in differentiation of a clone of Ly-1+ B cells.  

PubMed

CH12.LX, an in vitro subclone of a murine B cell lymphoma that makes IgM reactive with sheep erythrocytes (SRBC), has cell surface receptors for the lymphokine interleukin 2 (IL 2). The binding of recombinant murine IL 2 to these receptors did not stimulate CH12.LX cells to differentiate and secrete antibody. However, the binding of either of two monoclonal antibodies (Mab) specific for the IL 2 receptor increased the proportion of CH12.LX cells that secrete hemolytic IgM. The effect did not require the presence of antigen. One of the Mab, 3C7, is known to block the binding of IL2 to its receptor on T cells, whereas the other, 7D4, which also reacts with the IL 2 receptor, does not block the binding of IL 2. The differentiation of CH12.LX induced by 3C7, but not that induced by 7D4, was inhibited by recombinant IL 2. Neither IL 2 (up to 200 U/ml) nor 3C7 (up to 10 micrograms/ml) had any significant influence on incorporation of [3H]thymidine; 7D4 at 10 micrograms/ml decreased thymidine incorporation by about 60%. Mitomycin C and hydroxyurea, which both inhibit the incorporation of [3H]thymidine into CH12.LX cells, also both induce antibody secretion. In both cases, the concentration necessary to cause differentiation is substantially lower than that needed to cause detectable inhibition of thymidine uptake. We conclude that the IL 2 receptor on CH12.LX cells is a functional signal transducing molecule, and we discuss the possible inverse relationship between proliferation and differentiation. PMID:3106480

Bishop, G A; Haughton, G

1987-05-15

118

Low-Dose Interleukin-2 Therapy: A Driver of an Imbalance between Immune Tolerance and Autoimmunity  

PubMed Central

For many years, the role of interleukin-2 (IL-2) in autoimmune responses was established as a cytokine possessing strong pro-inflammatory activity. Studies of the past few years have changed our knowledge on IL-2 in autoimmune chronic inflammation, suggesting its protective role, when administered at low-doses. The disrupted balance between regulatory and effector T cells (Tregs and Teffs, respectively) is a characteristic of autoimmune diseases, and is dependent on homeostatic cytokines, including IL-2. Actually, inherent defects in the IL-2 signaling pathway and/or levels leading to Treg compromised function and numbers as well as Th17 expansion have been attributed to autoimmune disorders. In this review, we discuss the role of IL-2 in the pathogenesis of autoimmune diseases. In particular, we highlight the impact of the dysregulated IL-2 pathway on disruption of the Treg/Th17 balance, reversal of which appears to be a possible mechanism of the low-dose IL-2 treatment. The negative effects of IL-2 on the differentiation of follicular helper T cells (Tfh) and pathogenic Th17 cells, both of which contribute to autoimmunity, is emphasized in the paper as well. We also compare the current IL-2-based therapies of animal and human subjects with immune-mediated diseases aimed at boosting the Treg population, which is the most IL-2-dependent cell subset desirable for sufficient control of autoimmunity. New perspectives of therapeutic approaches focused on selective delivery of IL-2 to inflamed tissues, thus allowing local activity of IL-2 to be combined with its reduced systemic and pleiotropic toxicity, are also proposed in this paper. PMID:25322151

Kosmaczewska, Agata

2014-01-01

119

Induction with interleukin-2 antagonist for transplantation of kidneys from older deceased donors: an observational study  

PubMed Central

Background The most important limiting factor in kidney transplantation is the scarcity of donor organs. Consequently, there is an increased use worldwide of kidneys from older deceased donors. High donor age is a known risk factor for acute cellular rejection and premature graft failure, and the optimal immunosuppressive regimen in these circumstances remains to be established. Methods We investigated whether induction treatment with an interleukin 2 (IL-2) receptor antagonist improves graft survival and reduces rejection episodes in recipients of kidneys from deceased donors aged ? 60 years. Data were retrieved for all recipients transplanted at our center from 2004 to 2009 with a kidney from a deceased donor aged > 60 years. The outcome was compared between recipients treated with (IL-2 plus) or without (IL-2 minus) an IL-2 receptor antagonist. All recipients received a calcineurin inhibitor, steroids and mycophenolate. Results A total of 232 first-transplant recipients were included (IL-2 plus = 149, IL-2 minus = 83). IL-2 minus was associated with increased risk of early acute rejection (OR 2.42; 95% CI 1.25 to 4.68, P = 0.009) and steroid-resistant rejection (OR 8.04; 2.77 to 23.25, P< 0.001). IL-2 plus patients had superior two-year estimated uncensored (87% versus 70%, P = 0.001) and death-censored (95% versus 79%, P< 0.001) graft survival. Conclusions Induction treatment with IL-2 receptor antagonist was associated with a reduction in acute rejection episodes and improved two-year graft survival in patients transplanted with kidneys from older deceased donors. PMID:23799993

2013-01-01

120

Intratumoural and peripheral blood lymphocyte subsets in patients with metastatic renal cell carcinoma undergoing interleukin-2 based immunotherapy: association to objective response and survival  

Microsoft Academic Search

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-? and histamine. A total of

F Donskov; K M Bennedsgaard; H von der Maase; N Marcussen; R Fisker; J J Jensen; P Naredi; M Hokland

2002-01-01

121

Inadequate interleukin 2 production. A fundamental immunological deficiency in patients with major burns.  

PubMed Central

We studied the production of the two major mediators of cellular immune responses, Interleukin 1 (IL-1) and Interleukin 2 (IL-2), by the peripheral blood mononuclear cells of 23 burn patients (16 men, seven women, mean age 48.9 years) compared with 23 matched controls (16 men, seven women, mean age 46.7 years). Serial measurements were made of IL-1 production by adherent mononuclear cells after stimulation with lipopolysaccharide and of IL-2 production by lymphocytes after stimulation with phytohemagglutinin (PHA). Eighty determinations of IL-2 production by lymphocytes from 12 patients with greater than 30% body surface area burn revealed a mean IL-2 production of 0.71 u as compared with a mean of 1.23 u for patients with less than 30% burns (p = 0.04). Patients with greater than 30% body surface area burns had significantly reduced IL-2 production (p less than or equal to 0.05) until 60 days after injury, whereas those with smaller burns had reduced IL-2 production only at 20-29 and 30-39 days postburn. Nine burn patients with systemic sepsis showed significantly lower IL-2 production (p = 0.03) at 10-29 days postburn than nonseptic patients, and significantly less IL-2 production during septic episodes. Eight patients with greater than 50% suppression of lymphocyte response to PHA produced less IL-2 (0.4 u) than patients with less than 50% suppression, (1.07 u, p = 0.004). IL-1 production was significantly elevated as compared with controls (4.45 u vs. 3.6 u, p = 0.05) early after injury, but was subsequently within the normal range regardless of burn size. The percentage of circulating helper T-lymphocytes, the principal source of IL-2, was also reduced, although this did not always correlate with IL-2 production, which remained depressed after recovery of the helper T-cell population. These results indicate that failure to produce IL-2, a powerful mediator of cellular immune responses, is an important mechanism underlying the defective cell mediated immunity seen in burn patients. PMID:6331804

Wood, J J; Rodrick, M L; O'Mahony, J B; Palder, S B; Saporoschetz, I; D'Eon, P; Mannick, J A

1984-01-01

122

Interleukin-2 receptor (Tac antigen) expressed on adult T cell leukemia cells.  

PubMed

We studied the expression of the interleukin-2 (IL-2) receptor and the proliferative response to exogenous IL-2 of peripheral blood leukemic cells from patients with adult T cell leukemia (ATL) in order to see whether IL-2 receptor expressed on ATL cells is different from normal IL-2 receptor and whether it plays a role in the neoplastic growth in ATL. Peripheral blood leukemic cells from 42 patients with ATL examined expressed IL-2 receptors that were detected by anti-Tac monoclonal antibody when examined immediately after the separation of cells or after the culture for 24 or 48 h. The number of anti-Tac binding sites ranged from 3,100 to 11,400 in fresh cells and from 3,600 to 96,000/cell in short-term cultured leukemic cells, whereas phytohemagglutinin-P (PHA-P)-stimulated normal T cells exhibited 6,900-35,000 anti-Tac binding sites per cell. ATL-derived and human T cell leukemia/lymphoma virus, type I (HTLV-I)-infected cell lines such as MT-1 and Hut102 expressed a much higher number of anti-Tac binding sites. Leukemic cells from 15 patients with ATL examined showed no or very poor proliferative response to various concentrations of immunoaffinity-purified IL-2, although they expressed Tac antigen (Ag). Radiolabeled IL-2 binding experiments demonstrated that ATL leukemic cells could bind IL-2, and they expressed both high and low affinity IL-2 receptors, although the number of high affinity IL-2 receptor was much less than that of low affinity IL-2 receptor and that of anti-Tac binding sites. In contrast, leukemic T cells from a patient with T cell chronic lymphocytic leukemia (CLL), in whom HTLV-I infection was not demonstrated, responded as well as PHA-P-stimulated normal T cells, and their IL-2 receptors, unlike ATL cells, were modulated (down regulated) by anti-Tac antibody. No differences were noted between ATL cells and normal activated T cells in one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the IL-2 receptor. Thus, leukemic cells in ATL spontaneously and continuously express IL-2 receptor, which appears to be abnormally regulated and unresponsive to IL-2. These results, taken together with those on normal IL-2 receptors on HTLV-I-negative T-CLL cells, suggest that abnormal expression of the IL-2 receptor in ATL is closely associated with HTLV-I infection and may play a role in the neoplastic growth of ATL cells. PMID:2993359

Uchiyama, T; Hori, T; Tsudo, M; Wano, Y; Umadome, H; Tamori, S; Yodoi, J; Maeda, M; Sawami, H; Uchino, H

1985-08-01

123

Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2.  

PubMed Central

To study the effect of interleukin-2 (IL-2) on simian immunodeficiency virus (SIV) replication, pathogenesis, and immunogenicity, we replaced the nef gene of SIVmac239 by the IL-2 coding region. The virus, designated SIV-IL2, stably expressed high levels of IL-2 in cell culture. In comparison to SIVmac239, SIV-IL2 replicated more efficiently in peripheral blood mononuclear cells in the absence of exogenously added IL-2. To determine whether this growth advantage would be of relevance in vivo, four juvenile rhesus monkeys were infected with SIV-IL2 and four monkeys were infected with a nef deletion mutant of SIV (SIVdeltaNU). After a peak in the cell-associated viral load 2 weeks postinfection, the viruses could barely be isolated 3 to 7 months postinfection. Mean capsid antigen levels were higher in the SIV-IL2 group than in the nef deletion group 2 weeks postinfection. Viruses reisolated from the SIV-IL2-infected animals expressed high levels of IL-2 during the acute phase of infection. Deletions in the IL-2 coding region of SIV-IL2 were observed in two of the SIV-IL2-infected macaques 3 months postinfection. Urinary neopterin levels, a marker for unspecific immune stimulation, were higher in the SIV-IL2-infected macaques than in SIVdeltaNU-infected animals during the acute phase of infection. The SIV-specific T-cell-proliferative response and antibody titers were similar in both groups. Cytotoxic T cells directed against viral antigens were detected in all SIV-IL2-infected macaques and in two of the SIVdeltaNU-infected animals. Expression of IL-2 did not seem to alter the attenuated phenotype of nef deletion mutants fundamentally, although there might have been a slight increase in virus replication and immune stimulation during the acute phase of infection. Deletion of the viral IL-2 gene 3 months postinfection could be a consequence of a selective disadvantage due to local coexpression of viral antigen and IL-2 in the presence of an antiviral immune response. PMID:9032357

Gundlach, B R; Linhart, H; Dittmer, U; Sopper, S; Reiprich, S; Fuchs, D; Fleckenstein, B; Hunsmann, G; Stahl-Hennig, C; Uberla, K

1997-01-01

124

Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor.  

PubMed Central

Most biologic responses to IL-2 have been attributed to interaction of IL-2 with a high affinity receptor which consists of a heterodimer composed of two distinct IL-2-binding proteins (IL-2R alpha/IL-2R beta). However, both low affinity IL-2R alpha (55 kD) and intermediate affinity IL-2R beta (70-75 kD) also appear to be expressed independently on the cell surface. We investigated the receptor-specific regulatory effects of IL-2 on cytokine production in unstimulated and activated T cells. T cells were activated by stimulation of the antigen receptor complex with anti-CD3 mAb. IL-2 (10(2) U/ml, 1 nM) stimulation of resting cells resulted in a fivefold increase in GM-CSF release but in only minimal IFN-gamma release. IL-2 markedly augmented mRNA expression of GM-CSF but not IFN-gamma in unstimulated T cells. IL-2R beta mAb but not IL-2R alpha mAb decreased IL-2-induced GM-CSF release and mRNA expression from unstimulated T cells. IL-2 concentrations required for GM-CSF release from resting cells suggested ligand binding to an intermediate affinity receptor. GM-CSF and IFN-gamma release from activated T cells increased four- to fivefold in response to 1 nM IL-2 and IL-2 augmented both GM-CSF and IFN-gamma mRNA. IL-2R beta mAb but not IL-2R alpha mAb reduced GM-CSF release and mRNA expression in activated T cells stimulated with 1 nM IL-2. IL-2R alpha blockade markedly decreased IL-2-induced IFN-gamma release and mRNA expression from activated cells, while IL-2R beta blockade had little effect on IFN-gamma production in activated cells. IL-2R alpha blockade altered the affinity of the receptor mediating activated cell GM-CSF release from a high affinity to an intermediate affinity state. These studies indicate an independent role for IL-2R beta in mediating GM-CSF production from T cells. They also suggest that unstimulated and activated T cells, which express distinct IL-2 receptor moieties, mediate release of separate lymphokines and that different subunits of the IL-2 receptor may play an important role in the regulation of cytokine production. Images PMID:1828253

Burdach, S; Zessack, N; Dilloo, D; Shatsky, M; Thompson, D; Levitt, L

1991-01-01

125

The targeted expression of the human interleukin-2\\/interferon ?2b fused gene in ?-fetoprotein-expressing hepatocellular carcinoma cells  

Microsoft Academic Search

This study explores the use of a liver-specific albumin promoter and a tumor-specific ?-fetoprotein (AFP) enhancer to achieve\\u000a the regulated expression of the cytokine interleukin-2\\/interferon ?2b (IL-2\\/IFN?2b) fused gene for treatment of hepatocellular\\u000a carcinoma (HCC). The human AFP enhancer (E\\u000a \\u000a AFP\\u000a ) and albumin promoter (P\\u000a \\u000a ALB\\u000a ) were amplified from human chromosome DNA by the polymerase chain reaction. A

Ping He; Zhao-You Tang; Bin-Bin Liu; Sheng-Long Ye; Yin-Kun Liu

1999-01-01

126

JNK-Mediated BIM Phosphorylation Potentiates BAX-Dependent Apoptosis  

Microsoft Academic Search

Trophic factor deprivation (TFD) activates c-Jun N-terminal kinases (JNKs), culminating in coordinate AP1-dependent transactivation of the BH3-only BCL-2 proteins BIMEL and HRK, which in turn are critical for BAX-dependent cytochrome c release, caspase activation, and apoptosis. Here, we report that TFD caused not only induction but also phosphorylation of BIMEL. Mitochondrially localized JNKs but not upstream activators, like mixed-lineage kinases

Girish V Putcha; Siyuan Le; Stephan Frank; Cagri G Besirli; Kim Clark; Boyang Chu; Shari Alix; Richard J Youle; Art LaMarche; Anna C Maroney; Eugene M Johnson

2003-01-01

127

Cyclosporine inhibits expression of receptors for interleukin 2 and transferrin on mitogen-activated human T lymphocytes  

SciTech Connect

Cyclosporine (CyS) has been shown to inhibit activation of human T lymphocytes. A cascade of interactions involving Interleukin 2 (IL2), Interleukin 2 receptor (IL2R) and transferrin receptor (TR) is necessary for activation. In studies using peripheral blood mononuclear cells obtained from healthy donors, the authors measured the expression of IL2 and TR (using receptor-specific monoclonal antibodies and flow cytometry) and DNA synthesis (/sup 3/-thymidine incorporation) in response to PHA, OKT3, Leu 4 or Con A. In the presence of CyS (0.5 ..mu..g/ml) expression of IL2R and TR as well as DNA synthesis were markedly reduced. Upon serial dilutions of CyS, changes in DNA synthesis in response to OKT3 reflected changes in IL2R and TR levels, indicating all 3 parameters may be interrelated. Addition of exogenous IL2 partially abrogated the inhibitory effect of CyS on these activation parameters in response to PHA, OKT3 and Leu 4 but not to Con A. The authors results suggest that CyS is an effective inhibitor of mitogen-induced expression of IL2R and TR and that exogenous IL2 partially reverses this inhibitory effect.

John, J.K.; Prince, H.E.

1986-03-05

128

Expression levels and genetic polymorphisms of interleukin-2 and interleukin-10 as biomarkers of Graves’ disease  

PubMed Central

The aim of the present study was to determine whether the expression levels of interleukin (IL)-2 and IL-10 may be used as biological markers in Graves’ disease (GD) patients. A total of 256 individuals, including 118 GD patients and 138 healthy individuals, were enrolled into the study. Blood samples were collected from each patient and healthy individual, which were then subjected to enzyme-linked immunosorbent assay (ELISA). Total RNA and total proteins were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. In addition, restriction fragment length polymorphism (RFLP) analysis was performed to detect the presence of genetic polymorphisms. The ELISA results indicated that the IL-2 and IL-10 serum levels in the GD patients were increased by ~5.2 and ~7-fold when compared with the levels in the healthy controls. The results of RT-qPCR indicated that the mRNA expression levels of IL-2 and IL-10 were upregulated in the GD patients when compared with the healthy controls. Furthermore, the western blot analysis results revealed that the protein expression levels of IL-2 and IL-10 were significantly increased in the GD patients. RFLP analysis indicated that the increased number of GG single nucleotide polymorphisms (SNPs) in the GD group were detected in the ?330 locus of the IL-2 promoter and the ?1082 locus of the IL-10 promoter. In addition, the results indicated that the relatively high rates of homozygous GG SNPs (IL-2 ?330T/G and IL-10 ?1082A/G polymorphisms) on the alleles may be associated with the incidence of GD. The serum, mRNA and protein expression levels of IL-2 and IL-10 were significantly increased in GD patients when compared with the levels in the healthy controls. In conclusion, the expression levels and genetic polymorphisms of IL-2 and IL-10 may be potential biomarkers for the incidence of Graves’ disease in the population studied. PMID:25667655

LIANG, CUIGE; DU, WENHUA; DONG, QINGYU; LIU, XIAOMENG; LI, WENXIA; WANG, YUELI; GAO, GUANQI

2015-01-01

129

Differential glycosylation of interleukin 2, the molecular basis for the NOD Idd3 type 1 diabetes gene?  

PubMed

The insulin-dependent diabetes (Idd) gene, Idd3, has been localised to a 0.35 cM region of chromosome 3 containing the structural gene for the cytokine interleukin 2 (IL-2). While variation of the N-terminal amino acid sequence of IL-2 has been shown to correlate with Idd3 allelic variation, differences in induction of proliferation by IL-2 allotypes have not been detected. In the current study, we examined the electrophoretic migration of IL-2 allotypes and have found two distinct patterns, consistent with differences in glycosylation, that correlate with diabetes-resistance and susceptibility. These findings strongly suggest that IL-2 variants may be functionally distinct. PMID:10857762

Podolin, P L; Wilusz, M B; Cubbon, R M; Pajvani, U; Lord, C J; Todd, J A; Peterson, L B; Wicker, L S; Lyons, P A

2000-05-01

130

Diacylglycerol kinase-dependent formation of phosphatidic acid molecular species during interleukin-2 activation in CTLL-2 T-lymphocytes  

PubMed Central

Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species. PMID:23650609

Mizuno, Satoru; Sakai, Hiromichi; Saito, Masafumi; Kado, Sayaka; Sakane, Fumio

2012-01-01

131

The adaptor molecule TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating interleukin-2 receptor signaling  

PubMed Central

The number of Foxp3+ regulatory T (Treg) cells must be tightly controlled to efficiently suppress autoimmunity while not impairing normal immune responses. Here we show that the adapter molecule TRAF3 is intrinsically required for restraining lineage determination of thymic Treg cells. T cell-specific TRAF3 deficiency resulted in a 2-3 fold increase of Treg cell frequency, due to more efficient transition from T precursors to Foxp3+ Treg cells. TRAF3 dampened interleukin-2 (IL-2) signaling by facilitating recruitment of T cell protein tyrosine phosphatase (TCPTP) to the IL-2 receptor complex, resulting in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of Jak-STAT5 signaling. Our results identify a role for TRAF3 as an important negative regulator of IL-2 receptor signaling that impacts Treg cell development. PMID:25029551

Yi, Zuoan; Lin, Wai Wai; Stunz, Laura L.; Bishop, Gail A.

2014-01-01

132

A significant enhancement of therapeutic effect against hepatic metastases of M5076 in mice by a liposomal interleukin-2 (mixture).  

PubMed

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small hydrophobic liposomes under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 improved the distribution of IL-2 after intravenous administration as reported, previously. Liposomal IL-2 (300-10000 JRU/mouse per day) was significantly more effective than free IL-2 alone for inhibiting against the experimental metastases of M5076 in mice. The inhibitory effect of liposomal IL-2 was greatest in the liver. The ED(50) of liposomal IL-2 and that of free IL-2 in the liver were 1640 and 12500 JRU/mouse per day, respectively. This simple preparation (mixture) using IL-2 and liposome suspension is expected to have potential for increasing therapeutic efficacy against hepatic metastases. PMID:12175736

Kanaoka, Eri; Takahashi, Kouji; Yoshikawa, Takayoshi; Jizomoto, Hiroaki; Nishihara, Yoshitaka; Uchida, Naomi; Maekawa, Ryuji; Hirano, Koichiro

2002-08-21

133

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function  

SciTech Connect

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

Zhao Guohua [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Shi Lingfang [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Qiu Daoming [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Hu Hong [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Kao, Peter N. [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States)]. E-mail: peterkao@stanford.edu

2005-05-01

134

The role of endogenous interleukin-2 in proliferation of human carcinoma cell lines  

PubMed Central

Interleukin (IL-2) and IL-2R?/? have been shown to be expressed in human carcinomas in culture and in situ. Recently, expression of endogenous IL-2 and IL-2R in the cytoplasm was found to be up-regulated in tumour cells undergoing mitosis. This observation suggested that similar to its role in lymphocytes, the IL-2/IL-R pathway is involved in the regulation of carcinoma cell proliferation. Metabolic labelling followed by immunoprecipitation and Western blot results showed that IL-2 in carcinomas was identical to that in human lymphocytes. However, tumour cells did not secrete IL-2 detectable by immunoassays, although membrane-associated IL-2 was detectable on a proportion of these cells cultured in the absence of exogenous IL-2. Antibodies to IL-2 failed to inhibit proliferation of carcinoma cells, but antibodies specific for the ligand-binding site of the IL-2R were growth inhibitory. Growth of tumour cells was also inhibited by the immunosuppressive drugs, cyclosporin A (CsA), FK506 and rapamycin (RPA), known to interfere with the IL-2 pathway in lymphocytes. To further confirm the role of endogenous IL-2 in the growth of carcinomas, tumour cells were incubated with an IL-2-specific antisense oligonucleotide. The treatment was shown to transiently inhibit IL-2 mRNA and IL-2 protein expression as well as proliferation of tumour cells. Tumour cells treated with IL-2-specific antisense oligonucleotide demonstrated increased apoptosis in comparison to untreated or sense oligonucleotide-treated control cells. The data indicate that in human carcinomas, endogenous IL-2 promotes growth and protects tumour cells from apoptosis. © 1999 Cancer Research Campaign PMID:10555752

Reichert, T E; Kashii, Y; Stanson, J; Zeevi, A; Whiteside, T L

1999-01-01

135

[Massive apoptosis in P815 mastocytoma in vivo induced by interleukin 2 gene transduction].  

PubMed

A study was carried out to determine the mechanisms of the P815 murine mastocytoma rejection. IL2 gene was transferred into the P815 mastocytoma cells by the retroviral vector. The transduced cells were selected with G418 (1 mg/ml). The single P815/IL2 cells were obtained through the limit dilution method. Using digoxigenin-labelled IL2 cDNA as the probe, IL2 mRNA expression was detected by in situ hybridization. The activity of IL2 dependent cell line in the cultural medium of P815/IL2 cells was assayed by MTT Color reaction with 30-147 U/ml per 10(6) cells every 24 hours. The detection of the proliferation activity indicated that P815/IL2 cells grew slower than the parental cells. IL2 gene modified P815 mastocytoma cells were inoculated into DBA/2 mice. The results showed that parental P815 cells produced tumors in 100% DBA/2 mice about 5-6 days after injection and grew progressively, but P815/IL2 tumor cells did not grow at all or did much later and completely regressed after a transient growth in mice. The ultrastructural studies indicated that the P815/IL2 cells in vivo had changed remarkably. The heterochromatin was increased and nuclei became irregular in shape. Immature and mature special granules appeared near the Golgi's complexes. The most impressive findings were massive apoptosis in the tumor tissues. Massive apoptosis must be specific for IL2 gene transduction, because it was not found in tumors produced by the parental cells. Apopotic cells were phagocytosed by macrophages and nearby tumor cells. Various cell infiltration, composed predominantly of eosinophils and macrophages were seen in the tumor tissue. The results suggested that the difference in differentiation of P815/IL2 cells in vivo might be induced by factors from the host. Tumor rejection may be the result of the multi-cell-mediated reaction including eosinophils, macrophages and other host cells. PMID:9388873

Guo, N; Li, X; Liu, X

1996-10-01

136

Influence of Dopamine on the Altered Release of Prolactin, Luteinizing Hormone, and Follicle-Stimulating Hormone Induced by Interleukin2 in vitro  

Microsoft Academic Search

Interleukin-2 (IL-2) alters the release of anterior pituitary hormones at femtomolar concentrations from hemipituitaries incubated in vitro. This cytokine significantly lowered luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and stimulated prolactin (PRL) release, thus demonstrating a reciprocal action of the lymphokine on lactotrophs and gonadotrophs. Since dopamine (DA) is a powerful inhibitor of PRL release, in the present experiments we

Sharada Karanth; Umeko Marubayashi; Samuel M. McCann

1992-01-01

137

Maternal Mosaicism for a Novel Interleukin2 Receptor ?-Chain Mutation Causing X-Linked Severe CombinedImmunodeficiency in a Navajo Kindred  

Microsoft Academic Search

X-linked severe combined immunodeficiency disease (SCID) results from mutations of IL2RG, the gene encoding the interleukin-2 receptor ? chain, also known as the common ? chain (?c). A distinct form of autosomal recessive SCID occurs at an increased frequency among the Navajo Native American population, The disease gene responsible for autosomal Navajo SCID remains to be determined. We report the

Aengus S. O'Marcaigh; Jennifer M. Puck; Amy E. Pepper; Kenneth De Santes; Morton J. Cowan

1997-01-01

138

mRNA  

PubMed Central

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and “naked mRNA” for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters. PMID:23291946

Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-01-01

139

Molecular dissection of the interactions of an antitumor interleukin-2-derived mutein on a phage display-based platform.  

PubMed

A mutein with stronger antitumor activity and lower toxicity than wild-type human interleukin-2 (IL-2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL-2 receptor alpha chain (thought to be critical for both IL-2-driven expansion of T regulatory cells and IL-2-mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL-2-derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage-displayed IL-2 variants to induce proliferation of the IL-2-dependent CTLL-2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display-based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL-2 and related cytokines, accelerating the development of cytokine-derived therapeutics. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25683569

Rojas, Gertrudis; Carmenate, Tania; Leon, Kalet

2015-04-01

140

Treatment of metastatic renal cell carcinoma with subcutaneous interleukin 2: evidence for non-renal clearance of cytokines.  

PubMed Central

The circulating cytokine concentrations following administration of subcutaneous recombinant interleukin 2 (IL-2) in combination with interferon alpha and 5-fluorouracil used to treat advanced renal cancer were studied. One patient was anephric and on dialysis, and seven had normal biochemical renal function, although five had undergone single nephrectomy. The pharmacokinetics of IL-2 and changes in IL-6 and tumour necrosis factor (TNF)-alpha were essentially similar in all patients including the anephric patient, irrespective of the periods of dialysis, although at some time points, IL-2 concentrations were slightly higher in the anephric patient than in the others. These results show that for subcutaneous administration of low-dose IL-2, renal clearance of IL-2 is not important. This contrasts with high-dose, intravenous IL-2 where blood concentrations are higher and renal clearance seems to occur, perhaps because of saturation of the non-renal mechanisms of clearance. The subcutaneous route is certainly preferred if IL-2 is used in anephric patients and in those with impaired renal function, and it may be generally preferred for most purposes. PMID:9192992

Banks, R. E.; Forbes, M. A.; Hallam, S.; Jenkins, A.; Wadhwa, M.; Dilger, P.; Meager, A.; Thorpe, R.; Bowmer, C. J.; Joffe, J. K.; Patel, P.; Johnson, P. W.; Selby, P. J.

1997-01-01

141

Immunoglobulin production of human lymphocytes stimulated by Staphylococcus aureus Cowan I and pokeweed mitogen: differential effects of recombinant interleukin-2.  

PubMed Central

The number of immunoglobulin-secreting cells (ISC) upon stimulation with pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC) in recombinant interleukin-2 (rIL-2)-supplemented cultures of human peripheral blood mononuclear cells (PBMC) and co-cultures of B and T cells was studied. It has been shown that the addition of rIL-2 to culture can enhance or depress the number of ISC depending on the polyclonal B-cell activator used for culture stimulation. The SAC-induced response was enhanced in the presence of rIL-2, while the number of ISC in PWM-stimulated cultures was depressed. Moreover, in cultures stimulated simultaneously by both activators, the suppressive effect of rIL-2 prevailed, indicating that the reported direct effect of the lymphokine on SAC-activated B cells cannot overcome the suppressive activity of PWM-induced suppressor T cells. rIL-2 could not activate suppressor T cells in the absence of PWM, and has no effect on the number of helper or suppressor cells in the culture. PMID:2948904

Pryjma, J; Flad, H D; Ernst, M; Brandt, E; Ulmer, A J

1986-01-01

142

Epigenetic repression of interleukin 2 expression in senescent CD4+ T cells during chronic HIV type 1 infection.  

PubMed

The molecular mechanisms for IL2 gene-specific dysregulation during chronic human immunodeficiency virus type 1 (HIV-1) infection are unknown. Here, we investigated the role of DNA methylation in suppressing interleukin 2 (IL-2) expression in memory CD4(+) T cells during chronic HIV-1 infection. We observed that CpG sites in the IL2 promoter of CD4(+) T cells were fully methylated in naive CD4(+) T cells and significantly demethylated in the memory populations. Interestingly, we found that the memory cells that had a terminally differentiated phenotype and expressed CD57 had increased IL2 promoter methylation relative to less differentiated memory cells in healthy individuals. Importantly, early effector memory subsets from HIV-1-infected subjects expressed high levels of CD57 and were highly methylated at the IL2 locus. Furthermore, the increased CD57 expression on memory CD4(+) T cells was inversely correlated with IL-2 production. These data suggest that DNA methylation at the IL2 locus in CD4(+) T cells is coupled to immunosenescence and plays a critical role in the broad dysfunction that occurs in polyclonal T cells during HIV-1 infection. PMID:25001463

Nakayama-Hosoya, Kaori; Ishida, Takaomi; Youngblood, Ben; Nakamura, Hitomi; Hosoya, Noriaki; Koga, Michiko; Koibuchi, Tomohiko; Iwamoto, Aikichi; Kawana-Tachikawa, Ai

2015-01-01

143

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.  

PubMed Central

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans. Images PMID:1541678

Hibbs, J B; Westenfelder, C; Taintor, R; Vavrin, Z; Kablitz, C; Baranowski, R L; Ward, J H; Menlove, R L; McMurry, M P; Kushner, J P

1992-01-01

144

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.  

PubMed

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans. PMID:1541678

Hibbs, J B; Westenfelder, C; Taintor, R; Vavrin, Z; Kablitz, C; Baranowski, R L; Ward, J H; Menlove, R L; McMurry, M P; Kushner, J P

1992-03-01

145

Rat lymphoid cell lines producing human T cell leukemia virus. II. Constitutive expression of rat interleukin 2 receptor  

PubMed Central

Three rat lymphoid cell lines (TARS-1, TARL-2, and TART-1) (12) transformed by human T cell leukemia/lymphoma virus I (HTLV-I) had rearrangement of the beta chain gene of the T cell antigen receptor, and had integrated proviral DNA from HTLV-I in their genomes. As is the case with adult T cell leukemia (ATL)-derived human T cell lines transformed by HTLV-I, these rat cell lines unequivocally expressed interleukin 2 (IL-2) receptor, as determined by radiolabeled IL-2 binding. By Scatchard plot analysis, one of the cell lines, TART-1, proved to have high affinity receptors (Ka = 1.3 X 10(11)/M and 8.8 X 10(9)/M). Rat IL-2 receptor, not human IL-2 receptor, was expressed on HTLV+ rat cell lines, as demonstrated by the fact that they expressed antigens reactive with monoclonal antibodies (ART-18) against rat IL-2 receptor, but not with anti-Tac antibodies. The collective evidence indicates that the endogenous IL-2 receptor gene is activated in human and rat lymphoid cell lines with HTLV-I production. The mechanism of abnormal IL-2 receptor expression in HTLV infection is discussed. PMID:2985731

1985-01-01

146

Constitutive activation of the interleukin 2 gene in the induction of spontaneous in vitro transformation and tumorigenicity of T cells.  

PubMed Central

There is growing evidence to suggest that tumorigenic transformation of cells may result from aberrant regulation of autocrine growth factor production. In the current study we describe the spontaneous in vitro transformation of T-lymphocyte cell lines during routine cell culture as evidenced by autonomous growth without any requirement for stimulation or exogenous interleukin 2 (IL-2). These cells constitutively expressed the IL-2 gene and were inhibited from proliferating by addition of antibodies against IL-2, the IL-2 receptor, or IL-2 antisense oligonucleotides, thereby suggesting that the cell transformation resulted from IL-2-mediated autocrine growth. The transformed cells when injected into nude but not normal mice induced tumors that were inhibited by antibodies against IL-2 and the IL-2 receptor as well as by immunosuppressive drugs such as cyclosporin A. These studies demonstrate that aberrant regulation of IL-2 production can lead to spontaneous transformation of T cells in vitro, capable of inducing tumors in vivo. Our studies not only provide evidence for the important role played by autocrine growth factors in tumorigenicity but also stress the need to use caution while performing immunotherapy using in vitro-cultured T cells against cancer and viral infections, particularly in an immunodeficient host, to exclude any possible transfer of transformed mutant cells. Images PMID:8052634

Nagarkatti, M; Hassuneh, M; Seth, A; Manickasundari, K; Nagarkatti, P S

1994-01-01

147

Effect of various mouthwashes on the levels of interleukin-2 and interferon-gamma in chronic gingivitis.  

PubMed

The aim of this double blind study was to evaluate the effect of various mouthwashes: Chlorhexidine, Essential oil, Azadirachta indica (Neem) extract, and Povidone iodine on gingival tissue interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in patients with chronic gingivitis. A total of 8O patients (42 boys, 38 girls; mean age 16.0 +/- 1.8 years) were included in this study. Patients were randomly assigned into four groups of 20 each: Group I--Azadirachta indica (Neem) extract, Group II--Essential oil, Group III--Povidone iodine, and Group IV--Chlorhexidine. They were instructed to use these mouthwashes for two weeks. Plaque and gingival indices scores, and IL-2 and IFN-gamma levels in the gingival tissues were measured at baseline and after two weeks of mouthwash use. Results showed the reduction of plaque and gingival indices, and IL-2 and IFN-gamma level with Chlorhexidine, Essential oil, and Povidone iodine, which were found to be statistically significant. Although Neem reduced the level of plaque and gingival indices, and IL-2 and IFN-gamma to a certain level, it was not statistically significant. Therefore, Chlorhexidine, Essential oil, and Povidone iodine mouthwashes can be used as an adjunct to oral prophylaxis in reducing pro-inflammatory cytokines, IL-2 and IFN-gamma in patients with chronic gingivitis. PMID:18389675

Sharma, Shivalal; Saimbi, C S; Koirala, Bandana; Shukla, Rakesh

2008-01-01

148

In vivo cytokine production and recombinant interleukin 2 immunotherapy: an insight into the possible mechanisms underlying clinical responses.  

PubMed Central

Recombinant interleukin 2 (rIL-2), when given to patients with advanced malignant disease, induces a limited beneficial effect, with only 20-30% of patients with solid tumours responding. This present study has identified those patients with advanced colorectal cancer most likely to respond to rIL-2 therapy, by analysis of serum cytokine levels, prior to and during rIL-2 treatment, documented in responders and non-responders. Responders were found to have significantly lower pretreatment serum IL-6 and soluble IL-2 receptor levels (sIL-2R) than non-responders (P < 0.01 and P < 0.05 respectively). During rIL-2 infusion, responders developed high circulating levels of IL-6 and had low constant levels of prostaglandin E2 (PGE2). Non-responders failed to produce IL-6 and demonstrated elevated serum concentrations of PGE2, during infusions of rIL-2. Thus, an enhanced ongoing IL-6 and sIL-2R response, prior to therapy, was detrimental to subsequent treatment with rIL-2. Differential production and/or release of cytokines and prostaglandins, during therapy, further determined the likelihood of response to rIL-2. PMID:8198981

Deehan, D. J.; Heys, S. D.; Simpson, W. G.; Broom, J.; Franks, C.; Eremin, O.

1994-01-01

149

Regulatory dysfunction of the interleukin-2 receptor during HIV infection and the impact of triple combination therapy  

PubMed Central

The interleukin-2 (IL-2)/IL-2 receptor (IL-2R) system is the main regulatory determinant of T cell reactivity. Although it is well known that IL-2 secretion is impaired during HIV infection, up to now IL-2R expression has not been extensively studied in HIV-infected patients despite the use of IL-2 in clinical therapy trials. We show here that IL-2R expression in HIV patients with high viral load (group 1 in the study) is greatly enhanced on B lymphocytes, CD8 T lymphocytes, and monocytes, but not on CD4 T lymphocytes, compared with noninfected individuals. Paradoxically, this modified IL-2R expression does not lead to increased IL-2 responsiveness, except for B lymphocytes. In patients receiving triple combination therapy (TCT, two reverse transcriptase inhibitors and one protease inhibitor) that has triggered a drastic reduction in plasma viral load and an increase in CD4 counts (group 2 patients), IL-2R expression is significantly lower than in group 1 patients. Moreover, cells involved in cellular immunity and CD4 T lymphocytes have the capacity to respond to IL-2 after TCT. These results allow us to anticipate a beneficial role of IL-2 immunotherapy in combination with TCT. PMID:9736739

David, Denis; Bani, Lynda; Moreau, Jean-Louis; Treilhou, Marie-Pierre; Nakarai, Takayuki; Joussemet, Marcel; Ritz, Jerome; Dupont, Bertrand; Pialoux, Gilles; Thèze, Jacques

1998-01-01

150

Two mutational hotspots in the interleukin-2 receptor {gamma} chain gene causing human X-linked severe combined immunodeficiency  

SciTech Connect

Human severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immunity, is most commonly caused by mutations in the X-linked gene for interleukin-2 (IL-2) receptor {gamma} chain (IL2RG). For mutational analysis of IL2RG in males with SCID, SSCP screening was followed by DNA sequencing. Of 40 IL2RG mutations found in unrelated SCID patients, 6 were point mutations at the CpG dinucleotide at cDNA 690-691, encoding amino acid R226. This residue lies in the extracellular domain of the protein in a region not previously recognized to be significantly conserved in the cytokine receptor gene family, 11 amino acids upstream from the highly conserved WSXWS motif. Three additional instances of mutation at another CpG dinucleotide at cDNA 879 produced a premature termination signal in the intracellular domain of IL2RG, resulting in loss of the SH2-homologous intracellular domain known to be essential for signaling from the IL-2 receptor complex. Mutations at these two hotspots constitute >20% of the X-linked SCID mutations found by our group and a similar proportion of all reported IL2RG mutations. 41 refs., 5 figs., 1 tab.

Pepper, A.E.; Puck, J.M. [National Institutes of Health, Bethesda, MD (United States); Buckley, R.H. [and others

1995-09-01

151

Influence of immunomodulatory drugs on the cytotoxicity induced by monoclonal antibody 17-1A and interleukin-2.  

PubMed

Patients treated with monoclonal antibodies and cytokines for cancer receive often co-medication, which may influence treatment efficacy. Therefore, we investigated with a flowcytometric cytotoxicity assay the effect of several immunomodulatory drugs on antibody dependent cellular cytotoxicity (ADCC), interleukin-2 (IL-2) induced cytotoxicity and IL-2-induced-ADCC. We found that dexamethasone markedly inhibited the IL-2 induced cytotoxicity and the IL-2-induced-ADCC. Ondansetron, a 5-HT-3 serotonin receptor antagonist augmented significantly ADCC. Clemastine, a histamine type-2 receptor antagonist augmented the IL-2-induced-ADCC. The TNF antagonist thalidomide suppressed ADCC whereas pentoxifylline proved to be ineffective. Other tested drugs namely ibuprofen and indomethacin, both prostaglandin E2 antagonists, cimetidine a histamine type-2 receptor antagonist, the opioid pethidine, prostaglandin E2 and histamine exerted minor effects or had no influence on the tested parameters. We conclude that glucocorticosteroids should be avoided with monoclonal antibody and cytokine treatment. According to our in vitro data the other drugs tested did not have a negative impact on cellular cytotoxicity and ADCC. PMID:17562330

Flieger, Dimitri; Varvenne, Michael; Kleinschmidt, Rolf; Schmidt-Wolf, Ingo G H

2007-03-01

152

mRNA  

NSDL National Science Digital Library

Template for protein synthesis. Each set of three bases, called codons, specifies a certain protein in the sequence of amino acids that comprise the protein. The sequence of a strand of mRNA is based on the sequence of a complementary strand of DNA.

Darryl Leja (National Human Genome Research Institute REV)

2005-04-04

153

Rapid deterioration in quality of life during interleukin-2- and ?-interferon-based home therapy of renal cell carcinoma is associated with a good outcome  

Microsoft Academic Search

We conducted a prospective quality-of-life analysis during outpatient immunotherapy in 22 patients with progressive metastatic renal cell carcinoma (RCC) treated with subcutaneous interferon-?2a and subcutaneous interleukin-2. Patients' quality of life was assessed by the European Organization for Research and Treatment of Cancer quality-of-life questionnaire QLQ-C30 before (week 0) and once during immunotherapy (week 3). Advanced renal cancer patients completed a

J Atzpodien; Th Küchler; T Wandert; M Reitz

2003-01-01

154

Interleukin 2 Induces Tyrosine Phosphorylation and Activation of p72-74 Raf1 Kinase in a T-Cell Line  

Microsoft Academic Search

Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth

Bruce Turner; Ulf Rapp; Harald App; Mark Greene; Kunio Dobashi; John Reed

1991-01-01

155

A randomized Phase II study of interleukin-2 with and without beta-interferon for patients with advanced non-small cell lung cancer  

Microsoft Academic Search

Interleukin-2 (IL-2) and beta-interferon (?-IFN) are biologic agents with antitumor activity observed in preclinical models. Some studies of patients with advanced non-small cell lung cancer treated with IL-2 report relatively long survival, despite low response rates. Seventy-six evaluable patients with stage IV non-small cell lung cancer were treated in a randomized Phase II study with either IL-2 alone or IL-2

William J. Tester; Kyung Mann Kim; Robert L. Krigel; Philip D. Bonomi; John H. Glick; Robert F. Asbury; John M. Kirkwood; Ronald H. Blum; Joan H. Schiller

1999-01-01

156

Association of interleukin-2 and interferon-? production by blood mononuclear cells in infancy with parental allergy skin tests and with subsequent development of atopy  

Microsoft Academic Search

The mechanisms regulating the onset of atopic sensitization in human beings are not yet fully clarified. We assessed the capacity of mitogen-stimulated umbilical and peripheral blood mononuclear cells to produce interferon-? (IFN-?) and interleukin-2 (IL-2) at birth and at 9 months of age in 159 infants. Mononuclear cell production of both IFN-? and IL-2 at 9 months, but not at

Fernando D. Martinez; Debra A. Stern; Anne L. Wright; Catharine J. Holberg; Lynn M. Taussig; Marilyn Halonen

1995-01-01

157

Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes  

Microsoft Academic Search

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13

E. A. Grimm; A. Mazumder; H. Z. Zhang; S. A. Rosenberg

1982-01-01

158

Soluble Interleukin2 Receptor (sIL2R), Tumor Necrosis Factor Alpha (TNF-a) and Its Receptor as Prognostic Markers in \\  

Microsoft Academic Search

This study aimed at assessing whether pretreatment levels of interleukin-6 (IL6), interleukin-10 (IL- lo), tumor necrosis factor-a (TNF-a), soluble TNF type 1 receptor (p55-R-TNF) and soluble interleukin-2 receptor (sIL-2R) are related to known clinical and biological prognostic factors of lymphoma. Thirty-five patients diagnosed to have non-Hodgkin's lymphoma (NHL) were studied. Patients were treated by 6 cycles of multiagent chemotherapy regimen

Elham Abdel Samie; Sherif H Abdel Wahab; Amal E Khalifa

159

Production of interleukin-1? and interleukin-2 by separate, phenotypically different leukaemia and human T cell lymphotropic virus-1-transformed T cell clones  

Microsoft Academic Search

The relationship between interleukin-1 (IL-1) and interleukin-2 (IL-2) production and immunophenotype marker profiles was studied in a panel of 29 leukaemia and human T cell lymphotropic virus-l (HTLV-1)-transformed T cell lines. Culture supernatants from six of the 29 T cell lines tested increased IL-2 production by the MOLT-16 cell line in a manner similar to that of rIL-1? or rIL-1?.

V Holá?; J Minowada

1993-01-01

160

The release of interleukin-2 (IL2) and colony stimulating activity (CSA) in aplastic anemia patients: opposite behaviour with improvement of bone marrow function  

Microsoft Academic Search

Peripheral blood cells from patients with aplastic anemia were tested for their ability to release interleukin-2 (IL-2) and colony stimulating activity (CSA) before treatment. IL-2 release — as measured in the mouse thymocyte assay — was abnormally high in 18\\/34, and abnormally low in 10\\/34 patients. “Low” release was due to simultaneous release of thymocyte inhibitors. In 18 patients who

Catherine Nissen; Yolanda Moser; Johanna Weis; Andreas Wtirsch; Alois Gratwohl; Bruno Speck

1986-01-01

161

The effect of interleukin-2 on canine peripheral nerve sheath tumours after marginal surgical excision: a double-blind randomized study  

PubMed Central

Background The objective of this study was to evaluate the effect on outcomes of intraoperative recombinant human interleukin-2 injection after surgical resection of peripheral nerve sheath tumours. In this double-blind trial, 40 patients due to undergo surgical excision (<5 mm margins) of presumed peripheral nerve sheath tumours were randomized to receive intraoperative injection of interleukin-2 or placebo into the wound bed. Results There were no significant differences in any variable investigated or in median survival between the two groups. The median recurrence free interval was 874 days (range 48–2141 days), The recurrence-free interval and overall survival time were significantly longer in dogs that undergone the primary surgery by a specialist-certified surgeon compared to a referring veterinarian regardless of whether additional adjunct therapy was given. Conclusion Overall, marginal excision of peripheral nerve sheath tumours in dogs resulted in a long survival time, but adjuvant treatment with recombinant human interleukin-2 (rhIL-2) did not provide a survival advantage. PMID:23927575

2013-01-01

162

Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways.  

PubMed

Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease. PMID:25655587

Doucette, Carolyn D; Greenshields, Anna L; Liwski, Robert S; Hoskin, David W

2015-04-01

163

Interleukin-2 signalling is modulated by a labile disulfide bond in the CD132 chain of its receptor.  

PubMed

Certain disulfide bonds present in leucocyte membrane proteins are labile and can be reduced in inflammation. This can cause structural changes that result in downstream functional effects, for example, in integrin activation. Recent studies have shown that a wide range of membrane proteins have labile disulfide bonds including CD132, the common gamma chain of the receptors for several cytokines including interleukin-2 and interleukin-4 (IL-2 and IL-4). The Cys(183)-Cys(232) disulfide bond in mouse CD132 is susceptible to reduction by enzymes such as thioredoxin (TRX), gamma interferon-inducible lysosomal thiolreductase and protein disulfide isomerase, which are commonly secreted during immune activation. The Cys(183)-Cys(232) disulfide bond is also reduced in an in vivo lipopolysaccharide (LPS)-induced acute model of inflammation. Conditions that lead to the reduction of the Cys(183)-Cys(232) disulfide bond in CD132 inhibit proliferation of an IL-2-dependent T cell clone and concomitant inhibition of the STAT-5 signalling pathway. The same reducing conditions had no effect on the proliferation of an IL-2-independent T cell clone, nor did they reduce disulfide bonds in IL-2 itself. We postulate that reduction of the Cys(183)-Cys(232) disulfide in CD132 inhibits IL-2 binding to the receptor complex. Published data show that the Cys(183)-Cys(232) disulfide bond is exposed at the surface of CD132 and in close contact with IL-2 and IL-4 in their respective receptor complexes. In addition, mutants in these Cys residues in human CD132 lead to immunodeficiency and loss of IL-2 binding. These results have wider implications for the regulation of cytokine receptors in general, as their activity can be modulated by a 'redox regulator' mechanism caused by the changes in the redox environment that occur during inflammation and activation of the immune system. PMID:22645657

Metcalfe, Clive; Cresswell, Peter; Barclay, A Neil

2012-01-01

164

Differential effects of interleukin-2 and interleukin-4 on immunomodulatory role of platelet-activating factor in human B cells.  

PubMed Central

Platelet-activating factor (PAF), a naturally occurring phospholipid cytokine, is a potent mediator of allergic and inflammatory reactions, as well as a modulator of immune responses. In the present study we showed that PAF is involved in early B-cell activation, as demonstrated by the increased cyclic AMP (cAMP) generation by PAF in a time- and dose-dependent manner in anti-mu antibody- plus B-cell growth factor-activated normal human peripheral blood B lymphocytes. PAF also regulated differentiation by causing a biphasic response on immunoglobulin M (IgM) production with an inhibitory signal generated at 10(-6) M and a stimulatory signal generated at 10(-8) to 10(-10) M. PAF enhanced IgA secretion. The regulation exerted by PAF was shown to be specific because the addition of the PAR antagonist CV-3988 abrogated these effects and the inactive form of PAF, lyso-PAF, induced neither cAMP generation nor immunoglobulin secretion in normal human B cells. Other cytokines, interleukin-2 (IL-2) and IL-4, potent mediators of the immune response, were unable to elicit a cAMP response in B cells. However, the addition of PAF (10(-6) M) with wither IL-2 or IL-4 enhanced cAMP production above the levels enhanced by the addition of PAF alone. IL-2 or IL-4, individually, stimulated IgM production, yet costimulation with PAF resulted in a differential effect between IL-2 and IL-4. PAF down-regulated the IL-4-induced IgM secretion, whereas the IL-2-induced IgM secretion was enhanced. The presence of CV-3988 returned all valued to those obtained with IL-2 or IL-4 alone, demonstrating the specificity of PAF. These data suggest that PAF is an important B-cell immunomodulator which can interact with other leukocyte cell mediators. PMID:8556480

Patke, C L; Green, C G; Shearer, W T

1994-01-01

165

The 20th anniversary of interleukin-2 therapy: bimodal role explaining longstanding random induction of complete clinical responses  

PubMed Central

Background This year marks the twentieth anniversary of the approval by the US Food and Drug Administration of interleukin-2 (IL2) for use in cancer therapy, initially for renal cell carcinoma and later for melanoma. IL2 therapy for cancer has stood the test of time, with continued widespread use in Europe, parts of Asia, and the US. Clinical complete responses are variably reported at 5%–20% for advanced malignant melanoma and renal cell carcinoma, with strong durable responses and sustained long-term 5–10-year survival being typical if complete responses are generated. Methods The literature was reviewed for the actions and clinical effects of IL2 on subsets of T cells. The influence of IL2 on clinical efficacy was also sought. Results The review revealed that IL2 is capable of stimulating different populations of T cells in humans to induce either T effector or T regulatory responses. This apparent “functional paradox” has confounded a clear understanding of the mechanisms behind the clinical effects that are observed during and following administration of IL2 therapy. An average complete response rate of around 7% in small and large clinical trials using IL2 for advanced renal cell carcinoma and malignant melanoma has been shown from a recent review of the literature. Conclusion This review considers the published literature concerning the actions and emerging clinical effects of IL2 therapy, spanning its 20-year period in clinical use. It further details some of the recently described “bimodal” effects of IL2 to explain the apparent functional paradox, and how IL2 might be harnessed to emerge rapidly as a much more effective and predictable clinical agent in the near future. PMID:22904643

Coventry, Brendon J; Ashdown, Martin L

2012-01-01

166

Effects of subcutaneous interleukin-2 therapy on CD4 subsets and in vitro cytokine production in HIV+ subjects.  

PubMed Central

HIV infection is characterized by the reduction of the CD4+, CD45RA+, CD26+, and CD28+ lymphocyte subsets and of the in vitro production of IL-2, IL-4, and interferon-gamma; on the contrary, chemokine production is usually increased. These abnormalities are only partially restored by antiretroviral chemotherapy. Therapy with interleukin-2 has been proposed to restore the functions of the immune system, but the mechanisms by which IL-2 exerts its activities are unknown. The aim of this study was to define the effects of rIL-2 administration on CD4+, CD45RA+, CD45R0+, and CD26+ lymphocytes and on the in vitro production of IL-2, IL-4, IL-10, IFN-gamma, RANTES, and sCD30 in HIV+ patients. 10 HIV+ patients with CD4 cell counts between 200 and 500 cells/mm3 were treated with six cycles of subcutaneous recombinant IL-2 administration, in combination with zidovudine and didanosine. This therapeutic regimen resulted in a remarkable increase in the number of CD4+ cells and in the prolonged reduction of the levels of viremia. CD45R01 cells were expanded during the first cycle of therapy, while CD45RA+/CD26+ cells predominated after the third cycle. At this time, the in vitro production of IL-2, IL-4, IFN-gamma, and sCD30 were significantly upregulated. These results demonstrate that rIL-2 in HIV+ patients induces the reconstitution of the CD4/CD45RA lymphocytes subtype. This expanded cell population recovered the ability to produce in vitro IL-2, IL-4, and IFN-gamma. These effects may be beneficial to HIV+ patients by improving their immune response to microorganisms or vaccines. PMID:9389737

De Paoli, P; Zanussi, S; Simonelli, C; Bortolin, M T; D'Andrea, M; Crepaldi, C; Talamini, R; Comar, M; Giacca, M; Tirelli, U

1997-01-01

167

Efficacy of chemoimmunotherapy with cyclophosphamide, interleukin-2 and lymphokine activated killer cells in an intraperitoneal murine tumour model.  

PubMed Central

We have previously reported on the efficacy of intraperitoneal (i.p.) immunotherapy with interleukin-2 (IL-2) and adoptively transferred lymphokine activated killer (LAK) cells in an i.p. murine tumour model. Because of a dose-limiting toxicity associated with IL-2, cures are seldom observed. The development of treatment strategies that combine components that augment or synergise with the antitumour activity of IL-2 is crucial for the successful use of IL-2 in a clinical setting. Because of the known toxicity of high-dose IL-2 or high dose cyclophosphamide (CY) treatment, the goal of our experiments was to investigate the efficacy of chemoimmunotherapy with low or moderate doses of cyclophosphamide (CY) in combination with low or moderate doses of IL-2 with or without adoptively transferred LAK cells. Assessment of i.p. tumour growth 14 days after tumour inoculation, using the peritoneal cancer index (PCI) scoring system, demonstrated that combination treatment of established (day 3) i.p. tumour was clearly superior to single modality treatment. The effect was further enhanced by a second dose of CY at the end of a course of IL-2. Combination treatment led to a significant survival benefit. About 25% of the mice were cured, even when the dose of tumour cells at inoculation was increased. These experiments demonstrate the efficacy of combined treatment with IL-2, LAK cells and CY. Further research should be directed at the design of treatment schedules based on repetitive courses of chemoimmunotherapy associated with little toxicity. PMID:3264714

Eggermont, A. M.; Sugarbaker, P. H.

1988-01-01

168

Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo.  

PubMed Central

The effect of interleukin-2 (IL-2) on IL-4-induced IgE and IgG4 secretion by B cells in peripheral blood mononuclear cell (PBMC) preparations from non-atopic healthy humans and atopic dermatitis patients was investigated. PBMC were cultured at an optimal concentration of recombinant IL-4 with or without addition of IL-2 for 10 days. Native and recombinant IL-2 inhibited the IL-4-induced IgE and IgG4 secretion in a dose-dependent manner by cells from both normal and atopic donors. Rabbit antibodies to IL-2 or to the monoclonal anti-IL-2 receptor antibody anti-TAC reversed the IL-2 effect. Culturing cells with IL-4 and IL-2 for 1 or 2 days only slightly suppressed the IgE and IgG4 secretion whereas addition of IL-2 to IL-4 containing cultures on day 4 or 5 inhibited the IgE and IgG4 secretion more effectively. This is in contrast to interferon-gamma (IFN-gamma) which inhibited the IL-4 induced IgE and IgG4 secretion when added for the first 24 or 48 h but had no effect when added on days 4 or 5. The data demonstrate that both IL-2 and IFN-gamma act as antagonists in the IL-4-induced IgE and IgG4 secretion by human B cells; while IL-2 appears to inhibit relatively late in culture, IFN-gamma has an early inhibitory effect, suggesting that the two lymphokines inhibit the IL-4 effect by different mechanisms. PMID:1904325

Spiegelberg, H L; Falkoff, R J; O'Connor, R D; Beck, L

1991-01-01

169

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients.  

PubMed

Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases. PMID:25634360

Rosenzwajg, Michelle; Churlaud, Guillaume; Mallone, Roberto; Six, Adrien; Dérian, Nicolas; Chaara, Wahiba; Lorenzon, Roberta; Long, S Alice; Buckner, Jane H; Afonso, Georgia; Pham, Hang-Phuong; Hartemann, Agnès; Yu, Aixin; Pugliese, Alberto; Malek, Thomas R; Klatzmann, David

2015-04-01

170

Adoptive immunotherapy of human pancreatic cancer with lymphokine-activated killer cells and interleukin-2 in a nude mouse model  

SciTech Connect

A pancreatic cancer cell line was grown in orthotopic and heterotopic positions in young Swiss/NIH nude mice, which were tested with adoptive immunotherapy. Mice were injected with 1 x 10(7) human cancer cells in the subcutaneous tissue and duodenal lobe of the pancreas. The mice were randomly divided into four groups: group IA (LAK + IL-2) (N = 25) received 2 X 10(7) human lymphokine-activated killer (LAK) cells from normal donors by tail vein injection followed by 10,000 units of human recombinant interleukin-2 (IL-2) given intraperitoneally every 12 hours for 28 days; group IB (IL-2) (N = 27) was given the same dose of IL-2 alone; group IC (RPMI-1640) (N = 18) received a placebo consisting of 1 ml of RPMI-1640 intraperitoneally every 12 hours; and group ID (LAK) (N = 14) received 2 X 10(7) LAK cells but no IL-2. Toxicity was significantly higher in group IB, with a mortality rate of 45.5% (10/22 animals) versus a 0% mortality (0/25) in group IA. None of the group IA or IB animals died of pancreatic cancer during the experiment. The animals that did not receive IL-2 died before 28 days in 14.2% of group IC and in 16.7% of group ID. The area under the growth curve of subcutaneous tumors during the course of treatment and the pancreatic tumor weight at the end of treatment were compared in each group. Subcutaneous tumors had a reduced rate of growth in group IA animals compared to all the other treatments. Pancreatic tumor growth was slowed in group IA. The animals treated with IL-2 alone (group IB) showed some slowing of tumor growth that was intermediate between group IA, group IC, and group ID. A similar experiment was done with irradiated (375 rad) mice. Nine nude mice with tumors were treated with LAK + IL-2 (group IIA), eight received IL-2 alone (group IIB), and seven received placebo (group IIC).

Marincola, F.M.; Da Pozzo, L.F.; Drucker, B.J.; Holder, W.D. Jr. (Stanford Univ. School of Medicine, CA (USA))

1990-11-01

171

Cytochalasin-B-induced immunosuppression of murine allogeneic anti-tumor response and the effect of recombinant human interleukin-2.  

PubMed

Cytochalasin B (CB), administered i.p. to C57B1/6 mice in a single dose as a suspension in carboxymethylcellulose 2%/Tween 20 1%, inhibits in a dose-dependent and time-dependent manner the ability of spleen cells to respond to allogeneic P815 mastocytoma tumor cells in vitro. Spleen cells from CB-treated animals sensitized to X-irradiated P815 cells in 4-day cultures at a 50:1 responder:stimulator ratio and tested for specific cytotoxicity against 51Cr-labelled P815 target cells showed strong inhibition 3 h after CB treatment at a dose of 50 mg/kg. A dose of 25 mg/kg showed measureable but not statistically significant inhibition at 3 h, whereas 10 mg/kg produced only slight inhibition, and 5 mg/kg and 2 mg/kg were noninhibitory. None of the doses produced significant suppression 19 h or 72 h after CB treatment. Addition to the sensitization cultures of human recombinant interleukin-2 (rhIL-2) at 350 BRMP units/ml completely restored tumor lytic capacity. C57B1/1 mice treated with CB 50 mg/kg, i.p. and challenged i.p. with 3 x 10(7) allogeneic P815 mastocytoma cells showed a brief, time-dependent, statistically significant abrogation of allogeneic responsiveness consistent with transient reversible immunosuppression within 3-12 h following CB treatment. No such inhibition of host allogeneic responsiveness in vivo was observed when CB was administered 24 h prior to, simultaneously with, or 1, 2, or 4 days after tumor challenge. Thus CB at the highest tolerated i.p. dose in vivo causes only a transient inhibition of anti-allo-responsiveness measured in culture, and rhIL-2 used in vitro restores lytic capacity. The anti-allo effect of CB is also seen to be transient directly in vivo since allogeneic tumor outgrowth is permitted for only a brief period following administration of CB. These results indicate that the use of CB in vivo in anti-tumor chemotherapy protocols will not be complicated by profound or prolonged immunosuppressive effects. PMID:1901032

Bogyo, D; Fondy, S R; Finster, L; Fondy, C; Patil, S; Fondy, T P

1991-01-01

172

Relationship between Serum Soluble Interleukin-2 Receptor and Renal Allograft Rejection: A Hospital-Based Study in KashmirValley  

PubMed Central

Background: Even after adequate immunosuppression therapy, acute rejection continues to be the single most important cause of graft dysfunction after renal transplantation. Renal allograft biopsy continues to be the reference standard, though certain clinical and biochemical parameters are helpful in assessment of these patients. Renal allograft rejection is mediated by T lymphocytes, expressing cell surface interleukin-2 receptors (IL-2R) which has been suggested as a marker of acute rejection episodes after organ transplantation. Objective: To determine the pre- and post-transplantation serum soluble IL-2R levels in live related kidney transplant patients to predict acute rejection episodes. Methods: Serial serum samples from 75 recipients and 41 healthy controls were assessed for soluble IL-2R levels by ELISA. The outcome of the graft was also determined for each recipient. Results: The mean±SD serum soluble IL-2R levels in renal allograft recipients with rejection were significantly (p<0.001) higher than those without rejection (329.85±59.22 vs 18.12±11.22 pg/mL). The elevation of serum soluble IL-2R was evident in acute rejection episodes and found before elevation of serum creatinine. The higher values of serum soluble IL-2R in the rejection group were significantly reduced after recovery of allograft function by adequate anti-rejection therapy. 36.4% of patients in the rejection group had proven positive biopsies for the rejection and higher creatinine values, which was found to be statistically significant (p<0.001). A cohort of 41 healthy controls showed significantly (p<0.05) lower serum soluble IL-2R concentrations (15.27±7.79 pg/mL) when compared with the rejection group. Conclusion: Serum soluble IL-2R concentrations showed significant correlation with the acute rejection episodes in the renal allograft recipients. Prediction of soluble IL-2R levels might help the early detection of rejection episodes, which may pave way for the management of immunosuppression regimes and better graft functioning. PMID:25737772

Rasool, R.; Yousuf, Q.; Masoodi, K. Z.; Bhat, I. A.; A Shah, Z.; Wani, I. A.; Wani, M. S.

2015-01-01

173

Survival in renal cell carcinoma-a randomized evaluation of tamoxifen vs interleukin 2, alpha-interferon (leucocyte) and tamoxifen.  

PubMed Central

Metastatic renal cell carcinoma (RCC) has a poor prognosis. Conventional treatment strategies, including chemotherapy and hormonal therapy, have limited value. Although encouraging results have been achieved in terms of objective response using immunological manipulations, no conclusive studies yet exist with a controlled comparative evaluation of survival. Therefore, the present study was undertaken, which compared one of the present (and presumed best) treatments, interleukin 2/interferon-alpha (IL-2/IFN-alpha) and tamoxifen, with a control arm of tamoxifen only. Tamoxifen has been shown to potentiate in vivo anti-tumour activity of IL-2, and because of its non-toxic behaviour it was included in both groups. The study was open, randomized and included seven institutions in Sweden. The patients were stratified according to the different centres involved. An interim analysis was planned when a minimum of 100 patients were evaluable. The 128 patients finally included had a histologically documented metastatic RCC, with a life expectancy of more than 3 months, a performance status WHO 0-2 and no prior chemo- or immunotherapy. Informed consent was obtained from each patient. The patients randomized to the control arm (n = 63) received only tamoxifen 40 mg p.o. daily for at least 1 year or until progression. The patients (n = 65) randomized to biotherapy received subcutaneous recombinant IL-2, leucocyte IFN-alpha in a treatment cycle of 42 days, as well as tamoxifen p.o. In the absence of undue toxicity or disease progression, these patients received one additional treatment cycle of 42 days followed by maintenance treatment, consisting of 5 days therapy every 4 weeks, for 1 year, or until proven progression. Only two patients in the tamoxifen-only group received immunotherapy when the disease progressed, but without any beneficial effect. All patients received appropriate local treatment when indicated. The interim analysis demonstrated no survival advantage for either group, and therefore further inclusion of patients was stopped. The median follow-up was 11 months (range 0.4-48 months). The final survival analysis showed no significant differences between the two treatment arms in so far as comparison from the day of diagnosis of primary disease, from the day of first evidence of metastatic spread, or from the onset of treatment. This was valid both when the evaluation was performed with regard to intention to treat and when the analysis was directed only to patients that managed at least one treatment cycle (42 days) of IL-2/IFN-alpha. The adverse effects were more pronounced in the IL-2/IFN-alpha group. Although the number of patients is limited, the results raise doubt concerning immunotherapy with IL-2 and IFN-alpha as a routine treatment in the management of advanced RCC. The difference in cost of drugs and health care (drug costs per patient: IL-2/IFN-alpha $27000 vs tamoxifen $360) as well as adverse effects caused by IL-2/IFN-alpha are also factors of importance. The study emphasizes the need for more effort to find the 'optimal schedule' of immunotherapy, as well as the need for randomized controlled studies before approval of a new treatment in the routine setting. PMID:9579838

Henriksson, R.; Nilsson, S.; Colleen, S.; Wersäll, P.; Helsing, M.; Zimmerman, R.; Engman, K.

1998-01-01

174

Synthesis and Optimization of the Labeling Procedure of 99mTc-Hynic-Interleukin-2 for In vivo Imaging of Activated T lymphocytes  

PubMed Central

Introduction We have previously described the labeling of interleukin-2 (IL2) with 123I and 99mTc-N3S. Both radiopharmaceuticals were successfully applied in humans to image several inflammatory lesions and autoimmune diseases characterized by tissue infiltrating lymphocytes expressing the IL2 receptor (CD25). However, both radiopharmaceuticals had some specific disadvantages, such as cost and time of synthesis. Materials and methods Here, we describe a new improved method for labeling interleukin-2 with 99mTc using HYNIC-NHS and tricine as coligand. Several optimizations of reagent concentrations and labeling conditions were performed in order to standardize the procedure. After labeling, IL2 was purified by tC2 reverse-phase chromatography and tested in vitro and in vivo, in mice and in a normal volunteer. Results showed that this labeling procedure is cheap, fast, reliable, and reproducible, leading to a product with high specific activity (153 µCi/µg), high stability and capable of binding in vitro to CD25 positive cells. In vivo biodistribution in mice and human volunteer did not show any significant different from 99mTc-N3S-IL2. Conclusion In conclusion, the optimization of 99mTc-HYNIC-IL2 has a great advantage in terms of cost and time of production and a simple kit formulation can be considered for routine application to study patients affected by autoimmune diseases, graft rejection, or other chronic inflammatory disorders. PMID:19949980

D’Alessandria, Calogero; di Gialleonardo, Valentina; Chianelli, Marco; Mather, Stephen J.; de Vries, Erik F. J.; Scopinaro, Francesco; Dierck, Rudi A.

2009-01-01

175

Human immunodeficiency virus-infected adherent cell-derived inhibitory factor (p29) inhibits normal T cell proliferation through decreased expression of high affinity interleukin-2 receptors and production of interleukin-2.  

PubMed Central

Adherent cells from HIV-infected subjects as well as in vitro HIV-infected normal adherent cells produce spontaneously a 29-kD (p29) factor that inhibits mitogen-induced proliferation of normal T cells. p29 mediates a partial dose-dependent inhibition of total protein synthesis in both nonstimulated and PHA-activated cells that is associated with impaired PHA-induced expression of IL-2 receptor (IL-2R)alpha chain, HLA-class II molecules, and production of IL-2 by these cells; conversely, p29 does not modify the expression of IL-2R beta chain, 4F2, CD9, or transferrin receptor, or the production of IL-1 and TNF alpha by the cells. 1 h preincubation of the cells with p29 is sufficient to detect its biologic activity and added rIL-2 abrogates p29-induced inhibition of IL-2R alpha chain expression; however, p29 does not display any biologic effect on already expressed IL-2R alpha chains. The impaired expression of IL-2R alpha chain mediated by p29 is not due to a decreased accumulation of the corresponding mRNA transcripts, but is associated with a two-fold increase of intracellular cAMP. Binding experiments with 125I-rIL-2 reveals that p29 induces a 50% decrease in the number of both high and low affinity IL-2R per cell. p29 also inhibits alloantigen-induced proliferation of PBMC, whereas it does not modify IL-2-dependent proliferation of 48-h PHA-blasts that already express high affinity IL-2R. These findings indicate that p29 mediates its biologic activity during early stages of T cell activation affecting the expression of high affinity IL-2R and production of IL-2, through a nontranscriptional mechanism involving an increase of intracellular cAMP. Images PMID:1321845

Ammar, A; Sahraoui, Y; Tsapis, A; Bertoli, A M; Jasmin, C; Georgoulias, V

1992-01-01

176

Sequential immune monitoring in patients with melanoma and renal cell carcinoma treated with high-dose interleukin-2: immune patterns and correlation with outcome.  

PubMed

Interleukin-2 (IL-2) therapy leads to clinically relevant responses in 10-16 % of patients with metastatic melanoma (MMEL) or 10-30 % of patients with metastatic renal cell carcinoma (MRCC). To date, no biomarkers have been validated to identify patients who are likely to respond. We hypothesized that changes in T cell subset distribution in patients undergoing IL-2 therapy may correlate with treatment outcomes. Immune profiles of 64 patients (27-MMEL, 37-MRCC) were evaluated using flow cytometry at baseline, during (?three doses) and at the end of treatment cycle (30 ± 6 h after last dose), through two courses of IL-2 therapy. Changes in distribution and phenotype of circulating CD4 and CD8 lymphocyte subsets were compared (1) based on cancer types and (2) intra-patient during the course of the IL-2 therapy. Exploratory analysis of immunologic profiles was also performed based on treatment outcome. Independent of cancer type, IL-2 led to a transient decrease of circulating effector lymphocytes, while regulatory T cells gradually increased. Interleukin-2 differentially affected a subset of CD8 T cell expressing Foxp3, depending on malignancy type. In MMEL patients, IL-2 gradually expanded circulating CD8 Foxp3+ cells; in MRCC patients, IL-2 transiently increased expression of CD103 and CCR4 homing markers. Monitoring of adaptive immune variables early on and during the course of IL-2 therapy revealed transient alterations in immune profiles, specific to MMEL and MRCC patients, related to immune balance (and ultimately response to IL-2 therapy) or T cell egress from the circulation. PMID:25205170

Foureau, David M; Amin, Asim; White, Richard L; Anderson, William; Jones, Chase P; Sarantou, Terry; McKillop, Iain H; Salo, Jonathan C

2014-12-01

177

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.  

PubMed Central

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. PMID:8943338

Lécine, P; Algarté, M; Rameil, P; Beadling, C; Bucher, P; Nabholz, M; Imbert, J

1996-01-01

178

Immunogenicity of oral vaccination with Lactococcus lactis derived vaccine candidate antigen (UreB) of Helicobacter pylori fused with the human interleukin 2 as adjuvant.  

PubMed

Helicobacter pylori (H. pylori) infection remains a significant global public health problem. Vaccine, especially edible vaccine, is considered to be effective in the management of H. pylori infections. By using recombinant technology, Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. The aim of this study was to produce edible UreB (urease B) vaccine derived from L. lactis against H. pylori. The UreB subunit is the most effective and common immunogen of all strains of H. pylori. The UreB was produced as a chimeric protein fused with IL-2 (human interleukin 2) as the mucosal adjuvant. Mucosal immunization of mice with recombinant L. lactis NZ9000 containing the UreB-IL-2 protein elicited more anti-UreB antibody that specifically bounded to the purified bacterial UreB protein and more cytokines such as IFN-?, IL-4, and IL-17, and had a lower H. pylori burden and urease activity than control mice. These results suggest that the recombinant L. lactis expressing UreB-IL-2 can be potentially used as an edible vaccine for controlling H. pylori infection. PMID:24036137

Zhang, Hong-xin; Qiu, Yu-yu; Zhao, Ying-hui; Liu, Xin-ting; Liu, Ming; Yu, Ai-lian

2014-02-01

179

Role of interleukin-2 and interferon-gamma in inducing production of IgG subclasses in lymphocytes of human newborns.  

PubMed Central

Unlike lymphocytes from adults, lymphocytes from cord blood of neonates cannot synthesize immunoglobulin G (IgG) in response to pokeweed mitogen (PWM). By using this mitogen in concert with interferon-gamma (IFN-gamma), interleukin-2 (IL-2), or interleukin-6 (IL-6), we studied the induction of IgG subclass molecules in lymphocytes of human neonates. IFN-gamma induced a limited, but substantial, enhancement of IgG2 production by neonatal lymphocytes. IL-2 dose dependently increased the production of each neonatal IgG subclass, whereas IL-6 did not. However, in adult lymphocytes, and under specific conditions, IL-6 or IL-2 each increased the production of all four IgG subclasses. Early in the culture IFN-gamma synergized with IL-2 during the latter or whole culture period to enhance cord blood IgG2 levels. This finding contrasted with the adult IgG2 synthesis synergistically up-regulated by IFN-gamma and IL-6. IL-2 caused a graded increase in immunoglobulin production in neonatal lymphocytes with IgG3 being the highest and IgG2 the lowest, thus corresponding to the differential increase of serum levels of IgG3/IgG1 and IgG4/IgG2 early in childhood. Results suggest that IL-2, but not IL-6, is critical to the development of human IgG subclass production. PMID:8707348

Kawano, Y; Noma, T

1996-01-01

180

Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function  

SciTech Connect

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function.

Ades, E.W.; Hinson, A.; Butler, L.D.

1986-08-01

181

Effects of the serine analogues isoserine and serinol on interleukin-2 synthesis and phospholipid metabolism in a human T cell line Jurkat.  

PubMed

Phosphatidylserine has been implicated both in the regulation of protein kinase C activity and in the regulation of T lymphocyte activation. Taking into account the fact that some serine analogues modify the activity of the base exchange enzyme system responsible for the synthesis of phosphatidylserine and to a lesser extent the synthesis of phosphatidylcholine and phosphatidylethanolamine, in vitro, we have tested the ability of both isoserine and serinol to modify phospholipid synthesis in the T cell line Jurkat. It was found that serinol was able to decrease by 75% the amount of phosphatidylserine synthesized by the cells and also to decrease the synthesis of phosphatidylcholine and phosphatidylethanolamine, while phosphatidylinositol synthesis was not affected. Concomitantly, in serinol-treated Jurkat cells, interleukin-2 production was markedly inhibited. Monitoring the production of second messengers generated by T cell activators showed that in serinol-treated cells the production of diacylglycerol was impaired while Ca2+ mobilization remained unaffected. Serinol thus appeared to be a potential immunoregulatory molecule active at the level of protein kinase C regulation either through its interaction with phosphatidylserine or through diacylglycerol production. PMID:1768842

Pelassy, C; Mary, D; Aussel, C

1991-01-01

182

Analysis of liver lymphoid cell subsets pre- and post-in vivo administration of human recombinant interleukin 2 in a C57BL/6 murine system.  

PubMed

The systemic administration of high dose recombinant interleukin 2 (RIL-2) can mediate significant reductions in the number of hepatic metastases in a murine system. This effect is sensitive to host irradiation. Both large granular (LGLs) and small (SLs) lymphocytes have been implicated as the cells mediating the antitumor effect. Utilizing selective Percoll fractionation of liver nonparenchymal lymphoid cells, we have attempted to determine the cell types involved in tumor immunotherapy of murine liver metastases during RIL-2 administration. At a RIL-2 dose of 25,000 units given i.p. three times a day, the total number of lymphoid cells seen in murine livers reached a peak on day 6 after the onset of RIL-2 therapy, lasting until day 10 and ranging from 25 to 29 times baseline values. Both LGLs and SLs were identified and SLs made up over one-half the cells present in murine livers. Phenotypic analysis of LGLs and SLs revealed that during exposure to RIL-2, bands 5 + 6 SLs expressed the Thy-1.2, Lyt-2, and Lyt-1 antigens to a greater degree than LGLs. LGLs exposed to RIL-2 demonstrated a decrease in the expression of the asialo GM1 antigen during exposure to RIL-2; however, the 49H.8 antigen normally expressed on natural killer cells and not on circulating T-cells was found only on LGLs. The role of murine liver LGLs and SLs needs to be further characterized. PMID:2302721

Lafreniere, R; Borkenhagen, K; Bryant, L D; Anton, A R; Chung, A; Poon, M C

1990-03-01

183

Natural killer T cells constitutively expressing the interleukin-2 receptor ? chain early in life are primed to respond to lower antigenic stimulation  

PubMed Central

Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interlukin-2 receptor ? chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25-expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krüppel-like factor-2, a transcription factor associated with quiescent T cells, and require de novo T-cell receptor and CD28 co-stimulation to proliferate. In contrast to bona fide CD4/CD25-expressing regulatory T cells, neonatal iNKT cells do not suppress T-cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d-restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin-2 suggest that constitutive expression of CD25 ‘primes’ neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25-expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT-cell repertoire early in life. PMID:20545784

Ladd, Mihoko; Sharma, Ashish; Huang, Qing; Wang, Adele Y; Xu, Lixin; Genowati, Indira; Levings, Megan K; Lavoie, Pascal M

2010-01-01

184

A biochemotherapy regimen with concurrent administration of cisplatin, vinblastine, temozolomide (Temodal), interferon-alfa and interleukin-2 for metastatic melanoma: a phase II study.  

PubMed

Our objective was to evaluate the toxicity and antitumor efficacy of concurrent biochemotherapy in metastatic melanoma patients and the effectiveness of adding temozolomide to protect the brain from metastases. Twenty-three patients with advanced inoperable melanoma were hospitalized for 5-6 days for the following treatment: cisplatin 20 mg/m daily for 4 days, vinblastine 1.6 mg/m daily for 4 days and oral temozolomide 250 mg/m daily for 5 days, with 18 x 10 IU/m intravenous interleukin-2 by continuous infusion for 4 days (the dose was cut daily by 50%) and 5 x 10 U/m interferon-alfa subcutaneously daily for 5 days, repeated at 28-day intervals for a maximum of nine courses. According to the standard World Health Organization response criterion, the objective response rate was 43.4% and the median survival was 18.6 months. All but one patient survived for more than 12 months, and no responding patient progressed first in the brain. Substituting dacarbazine by temozolomide in the MD Anderson melanoma section protocol appears to offer protection against dissemination of brain metastases, equal activity in the periphery and a possible lower incidence of toxicity due to the oral route. PMID:16432458

Ron, Ilan G; Sarid, David; Ryvo, Larisa; Sapir, Einat Even; Schneebaum, Shlomo; Metser, Ur; Asna, Noam; Inbar, Moshe J; Safra, Tamar

2006-02-01

185

Evidence for a Structural Motif in Toxins and Interleukin-2 That May Be Responsible for Binding to Endothelial Cells and Initiating Vascular Leak Syndrome  

NASA Astrophysics Data System (ADS)

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Baluna, Roxana; Rizo, Josep; Gordon, Brian E.; Ghetie, Victor; Vitetta, Ellen S.

1999-03-01

186

The prognostic significance of stable disease following high-dose interleukin-2 (IL-2) treatment in patients with metastatic melanoma and renal cell carcinoma.  

PubMed

High-dose interleukin-2 (HD IL-2) is an approved immunotherapy agent for metastatic melanoma and renal cell carcinoma resulting in objective responses in 15-20 % of patients. An additional subset of patients achieves stable disease, and the natural history of these patients has not been well documented. We hypothesized that stable disease following HD IL-2 is associated with a survival advantage. To explore this hypothesis, a retrospective chart review of 305 patients diagnosed with metastatic melanoma or renal cell carcinoma treated with HD IL-2 was conducted. Patient characteristics, response based on standard RECIST criteria and overall survival were analyzed using the Kaplan-Meier method and associations with clinical response were compared using a log-rank test. Two hundred and forty-five patients had melanoma and 60 had renal cell carcinoma. Of these, 217 had complete data available for analysis. Fifty-nine percentage had progressive disease (PD), 26 % had stable disease (SD) and 15 % had an objective complete (CR) or partial response (PR). Median overall survival was 16.8 months for all patients with available survival data; patients with PD had a median survival of 7.9 months compared to 38.2 months for stable disease, while the median has not been reached for those with objective responses. This retrospective data support an association between overall survival and stable disease, suggesting that clinical benefit may be underestimated for patients treated with HD IL-2. The data further support the use of disease control rate (CR + PR + SD) as a more meaningful endpoint for future clinical studies of tumor immunotherapy, including future studies of HD IL-2. PMID:25603775

Hughes, Tasha; Klairmont, Matthew; Broucek, Joseph; Iodice, Gail; Basu, Sanjib; Kaufman, Howard L

2015-04-01

187

The targeted expression of the human interleukin-2/interferon alpha2b fused gene in alpha-fetoprotein-expressing hepatocellular carcinoma cells.  

PubMed

This study explores the use of a liver-specific albumin promoter and a tumor-specific alpha-fetoprotein (AFP) enhancer to achieve the regulated expression of the cytokine interleukin-2/interferon alpha2b (IL-2/IFNalpha2b) fused gene for treatment of hepatocellular carcinoma (HCC). The human AFP enhancer (E(AFP)) and albumin promoter (P(ALB)) were amplified from human chromosome DNA by the polymerase chain reaction. A recombinant retrovirus was constructed including, as a selectable marker, the neoR gene and the IL-2/IFNalpha2b fused gene controlled by E(AFP)-P(ALB). The liver-targeted expression pattern of the IL-2/IFNalpha2b fused gene was observed when this product was tested in the culture medium of the infected cells (IL-2 activity was 850 IU/10(6) cells, IFNalpha activity was 320 IU/10(6) cells). Moreover, The growth of the IL-2/IFNalpha2b-fused-gene-infected HCC cells, SMMC7721, was clearly suppressed by the second week after innoculation of nude mice compared to the control SMMC7721 cells infected with LXSN and untreated SMMC7721 cells (0.5 +/- 0.1 cm versus 1.4 +/- 0.2 cm and 1.6 +/- 0.2 cm, P < 0.05). The results showed that the combined transcriptional regulatory sequences of E(AFP)-P(ALB) could control the targeted expression of cytokine genes in AFP-positive human HCC cells, and the expression level of the IL-2/IFNalpha2b fused gene was positively correlated to the level of AFP expression in the infected cells. The IL-2/IFNalpha2b fused protein that was expressed has the functions of both IL-2 and IFNalpha. Therefore, this study illustrates the superiority of using transcriptionally targeted recombinant retrovirus vectors in cytokine-based gene therapy. PMID:10190313

He, P; Tang, Z Y; Liu, B B; Ye, S L; Liu, Y K

1999-01-01

188

An adenoviral vector expressing functional heterogeneous proteins herpes simplex viral thymidine kinase and human interleukin-2 has enhanced in vivo antitumor activity against medullary thyroid carcinoma.  

PubMed

To explore a more efficient multi-gene antitumor treatment, we developed an adenoviral vector expressing both herpes simplex virus thymidine kinase (HSVtk) and human interleukin-2 (hIL-2) (AdCMVTKhIL2). Production of hIL-2 is expected to augment antitumor T cell and natural killer cell activity. Two separate cassettes expressing HSVtk and hIL-2, each under the control of the human cytomegalovirus (CMV) immediate early gene promoter, were inserted into the early 1 region of adenovirus type 5. This vector showed similar direct cytotoxicity towards infected rat medullary thyroid carcinoma (rMTC) cells as did the single gene vector, AdCMVtk. rMTC cells infected with the virus in vitro showed high sensitivity to ganciclovir. After infection with AdCMVTKhIL2 at 100 m.o.i. for 1 h, more than 20 000 U hIL-2 were produced during 24 h by 1 × 10(6) rMTC cells on day 2 and day 3. hIL-2 was also detected in the supernatants of primary cultures from tumors treated in vivo by the AdCMVTKhIL2 vector. Infected cells lost their tumorigenicity when transplanted subcutaneously into syngeneic rats, whereas all control animals developed tumors. More than 63% of tumors (19 out of 30 treated tumors) were destroyed when AdCMVTKhIL2 was injected intratumorally, compared with 38% when tumors were treated with AdCMVIL2, and 12% when treated with AdCMVtk, indicating an antitumor effect superior to that of each single vector given alone at the same dosage. These results indicate that the AdCMVTKhIL2 vector efficiently produces both HSVtk and hIL-2, and provides an enhanced antitumor activity. PMID:11733228

Zhang, R; DeGroot, L J

2001-12-01

189

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma.  

PubMed

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cell engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8+ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4+ T lymphocytes were scarce. Our results suggest that the CD8+ T lymphocytes are implicated in the anti-tumour reaction elicited by the IL-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC. PMID:8954146

Lausson, S; Fournes, B; Borrel, C; Milhaud, G; Treilhou-Lahille, F

1996-10-01

190

Enhancement of human lymphocyte proliferative response to purified protein derivative by an anti-interleukin-2 receptor alpha chain antibody (CD25).  

PubMed Central

While it is clear that the beta subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2-induced signal transduction, the function of the alpha subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between IL-2 and the IL-2R alpha subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2R alpha antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [3H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.2-6.2-fold) on the second day after maximal [3H]thymidine uptake had occurred. By itself, CD25-8D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2R alpha antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2R alpha chain plays a negative regulatory role in signal transduction. PMID:7821963

Kasinrerk, W; Majdic, O; Praputpittaya, K; Sittisombut, N

1994-01-01

191

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.  

PubMed Central

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha. Images Fig. 1 Fig. 2 Fig. 4 PMID:7724529

Richardson, J H; Sodroski, J G; Waldmann, T A; Marasco, W A

1995-01-01

192

Characterization of human interleukin 2 receptor (Tac antigen) in normal and leukemic T cells: co-expression of normal and aberrant receptors on Hut-102 cells.  

PubMed

Human interleukin 2 receptors ( IL2R ) from various cell sources such as mitogen-activated normal human T cells, adult T cell leukemia (ATL)-derived cell line cells (MT-1, ATL-6, Hut-102), and a natural killer-like cell line YT cells, were studied both by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with a monoclonal anti- IL2R antibody (anti-Tac). In synthetic labeling with [35S]methionine, anti-Tac specifically precipitated two glycoproteins, one with m.w. of 60,000 to 65,000 and pI 4.2 to 4.7, and the other with m.w. about 40,000 and pI 6.2 to 6.5. A study of surface iodination revealed that the former component was membrane-associated mature IL2R , and a pulse-chase study showed that the latter component was a precursor for IL2R that already possessed tunicamycin-sensitive N-linked sugar side chain(s). This precursor was found to be posttranslationally processed into mature IL2R by further glycosylation within 240 min. Among the cells studied, a human T cell leukemia virus-positive leukemic cell line, Hut-102, was distinctive in that it possessed an additional membrane glycoprotein (m.w. 55,000 to 60,000, pI 4.2 to 5.0) also defined by anti-Tac. This unique glycoprotein of Hut-102 cells appeared to be an aberrant IL2R . PMID:6327813

Wano, Y; Uchiyama, T; Fukui, K; Maeda, M; Uchino, H; Yodoi, J

1984-06-01

193

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.  

PubMed Central

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity. PMID:9199305

Lin, J X; Leonard, W J

1997-01-01

194

A phase I study of high-dose interleukin-2 with sorafenib in patients with metastatic renal cell carcinoma and melanoma.  

PubMed

This study was designed to evaluate the safety and feasibility of high-dose interleukin-2 (HD IL-2) followed by sorafenib in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC). Biomarkers relevant to the antitumor effects of IL-2 that may be altered by sorafenib including the percentages of natural T-regulatory cells (Tregs), myeloid-derived suppressor cells (MDSC), and STAT5 phosphorylation (pSTAT5) in T cells were evaluated. We hypothesized that the proposed treatment schedule is feasible and safe and may lead to enhanced tumor response. A phase I dose escalation trial was conducted in patients with either metastatic RCC or MM. HD IL-2 (600,000 IU/kg IV q8h × 8-12 doses) was administered on days 1-5 and 15-19, followed by sorafenib on days 29-82. The sorafenib dose was escalated. The percentage of Tregs, MDSC, and pSTAT5 in T cells were evaluated in peripheral blood by flow cytometry. Twelve of the 18 patients were evaluable for dose-limiting toxicity. No dose-limiting toxicity was observed. The treatment-related toxicity was predictable and did not seem to be additive with this schedule of administration. Partial responses were seen in 3 patients. No significant changes in the percentage of circulating Treg and MDSC were observed, whereas sorafenib did not adversely affect the ability of IL-2 to induce pSTAT5 in T cells. HD IL-2 followed by sorafenib was safe and feasible in patients with MM and RCC and did not adversely affect T-cell signaling through STAT5 in response to IL-2. PMID:24598448

Monk, Paul; Lam, Elaine; Mortazavi, Amir; Kendra, Kari; Lesinski, Gregory B; Mace, Thomas A; Geyer, Susan; Carson, William E; Tahiri, Sanaa; Bhinder, Arvinder; Clinton, Steven K; Olencki, Thomas

2014-04-01

195

Inhibition of caspase or FADD function blocks proliferation but not MAP kinase-activation and interleukin-2-production during primary stimulation of T cells.  

PubMed

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. PMID:12115619

Mack, Astrid; Häcker, Georg

2002-07-01

196

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.  

PubMed

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2R?) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2R? abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2R? in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2R? completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2R? interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2R? contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2R?, indicating IL2R? and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2R? in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.Oncogene advance online publication, 11 August 2014; doi:10.1038/onc.2014.243. PMID:25109334

Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

2014-08-11

197

Phase I trial of biochemotherapy with cisplatin, temozolomide, and dose escalation of nab-paclitaxel combined with interleukin-2 and interferon-? in patients with metastatic melanoma  

PubMed Central

The primary objective of this study was to determine the safety, toxicity, and maximum tolerated dose of nanoparticle albumin-bound (nab)-paclitaxel as part of biochemotherapy for metastatic melanoma and to determine whether substituting nab-paclitaxel for less potent agents could increase response rates and duration. Treatment consisted of intravenous cisplatin (20 mg/m2) on days 1–4, oral temozolomide (250 mg/m2) on days 1–3, subcutaneous interferon-? (5 × 106 IU/m2) on days 1–5, and continuous intravenous interleukin-2 (9 × 106 IU/m2) for 96 h on days 1–4. A standard 3 + 3 dose escalation method was used; the nab-paclitaxel starting dose was 100 mg/m2 on day 1 and 70 mg/m2 on day 5. The treatment cycle was repeated every 3 weeks and toxicity was assessed weekly. Ten patients were enrolled. Dose-limiting toxicities included diarrhea, transaminasemia, and neutropenia. The maximum tolerated dose was not identified because the nab-paclitaxel dose on day 1 at the lowest planned dose (80 mg/m2) caused dose-limiting toxicity in two of five patients. Of the nine patients who were evaluable for response, five had a partial response. The median time to disease progression was 5.30 months and the median overall survival was 8.73 months. Six patients developed central nervous system metastasis at a median of 5.33 months after treatment initiation. Biochemotherapy including nab-paclitaxel according to the doses and schedule regimen used in the present study has significant toxicity. Substituting dacarbazine with temozolomide did not prevent central nervous system metastasis in patients with metastatic melanoma. PMID:24743052

Alrwas, Anas; Papadopoulos, Nicholas E.; Cain, Suzanne; Patel, Sapna P.; Kim, Kevin B.; Deburr, Tawania L.; Bassett, Roland; Hwu, Wen-Jen; Bedikian, Agop Y.; Davies, Michael A.; Woodman, Scott E.; Hwu, Patrick

2014-01-01

198

Pilot trial of interleukin-2 and zoledronic acid to augment ?? T cells as treatment for patients with refractory renal cell carcinoma  

PubMed Central

Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human V?9 V?2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these V?9 V?2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of V?9 V?2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in V?9 V?2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of V?9 V?2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of V?9 V?2 T cell in patients with metastatic RCC. PMID:21647691

Lang, Joshua M.; Kaikobad, Mahazarin R.; Wallace, Marianne; Staab, Mary Jane; Horvath, Dorothea L.; Wilding, George; Liu, Glenn; Eickhoff, Jens C.; Malkovsky, Miroslav

2011-01-01

199

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694.  

PubMed

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases. PMID:25593320

Zhong, Yiming; Dong, Shuai; Strattan, Ethan; Ren, Li; Butchar, Jonathan P; Thornton, Kelsey; Mishra, Anjali; Porcu, Pierluigi; Bradshaw, J Michael; Bisconte, Angelina; Owens, Timothy D; Verner, Erik; Brameld, Ken A; Funk, Jens Oliver; Hill, Ronald J; Johnson, Amy J; Dubovsky, Jason A

2015-03-01

200

Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells  

PubMed Central

The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. PMID:25552553

Ramphul, Urvashi N.; Garver, Lindsey S.; Molina-Cruz, Alvaro; Canepa, Gaspar E.; Barillas-Mury, Carolina

2015-01-01

201

Targeting of TAK1 by the NF-B protein Relish regulates the JNK-mediated  

E-print Network

in genetically tractable inverte- brates, especially Drosophila melanogaster, have con- tributed greatly to our infection. Genetic screens for Drosophila mu- tants susceptible to pathogenic challenge have identified two in Drosophila Jin Mo Park,1 Helen Brady,3 Maria Grazia Ruocco,1 Huaiyu Sun,2 DeeAnn Williams,2 Susan J. Lee,2

202

mRNA stability in mammalian cells.  

PubMed Central

This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

Ross, J

1995-01-01

203

Durable responses and reversible toxicity of high-dose interleukin-2 treatment of melanoma and renal cancer in a Community Hospital Biotherapy Program  

PubMed Central

Background High-dose interleukin-2 (IL-2) has been FDA-approved for over 20 years, but it is offered only at a small number of centers with expertise in its administration. We analyzed the outcomes of patients receiving high-dose IL-2 in relation to the severity of toxicity to ascertain if response or survival were adversely affected. Methods A retrospective analysis of the outcomes of 500 patients with metastatic renal cell carcinoma (RCC) (n?=?186) or melanoma (n?=?314) treated with high-dose IL-2 between 1997 and 2012 at Providence Cancer Center was performed. IL-2 was administered at a dose of 600,000 international units per kg by IV bolus every 8 hours for up to 14 doses. A second cycle was administered 16 days after the first and patients with tumor regression could receive additional cycles. Survival and anti-tumor response were analyzed by diagnosis, severity of toxicity, number of IL-2 cycles and subsequent therapy. Results The objective response rate in melanoma was 28% (complete 12% and partial 16%), and in RCC was 24% (complete 7% and partial 17%). The 1-, 2- and 3-year survivals were 59%, 41% and 31%, for melanoma and 75%, 56% and 44%, for RCC, respectively. The proportion of patients with complete or partial response in both melanoma and RCC was higher in patients who a) required higher phenylephrine doses to treat hypotension (p?100,000 platelets; p?

2014-01-01

204

Deficiency of interleukin-2 activity upon addition of soluble egg antigen to cultures of spleen cells from mice infected with Schistosoma japonicum.  

PubMed Central

Schistosoma japonicum-infected C57BL/6 mice show similar dynamics of hepatic granulomatous inflammation (HGI) and delayed hypersensitivity (DH) elicited by soluble egg antigens (SEA) which reach peak levels at 9 weeks of infection and then spontaneously regress. The in vitro SEA-induced proliferation of spleen cells (SC) from infected animals attained its high point and then declined when SC from 5-week-infected mice were used. The present study determined the dynamics of interleukin-2 (IL-2) production by SEA-challenged SC from infected mice in an attempt to link the level of IL-2 production to the spontaneous regression of the aforementioned T-cell-mediated immune responses. The production of IL-2 by SEA-stimulated SC reached its peak when cells from 7-week-infected mice were challenged at least 2 weeks after the peak of the proliferative response, but declined at about the same time as the HGI and DH responses. Therefore, the decline in IL-2 activity cannot alone explain the diminished proliferative response but could account for the reduction in HGI and DH in vivo. Some possible mechanisms that might explain the IL-2 deficiency were examined. This deficiency is not due to the in vitro binding of IL-2 by the SC of infected mice and is, therefore, likely to be due to underproduction of IL-2. Nor is the deficiency explained by reduced numbers of antigen-presenting cells (macrophages and B cells) or of L3T4+ T lymphocytes or by suppression of IL-2 production by macrophages or macrophage products such as prostaglandins. However, suppression of IL-2 production was observed consistently upon coculture of SC from acutely infected mice with SC from mice infected for 10 weeks. The cells which suppress appear to be Lyt2+ T cells. The data are consistent with the hypothesis that suppressor T cells inhibit the production of IL-2 and perhaps of other cytokines or lymphokines and that this suppression explains the spontaneous down-regulation of HGI which occurs during schistosomiasis japonica. PMID:2968321

Stavitsky, A B; Harold, W W

1988-01-01

205

Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription.  

PubMed Central

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function. To define further both the specificity of CsA and the level at which it interferes with lymphokine gene expression, we have studied its effects on TCGF mRNA accumulation as well as TCGF gene transcription. These studies were performed with a cloned human leukemic T-cell line (Jurkat, subclone 32), which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of TCGF. In these cells, high levels of TCGF mRNA were present in induced but not in uninduced Jurkat cells as judged by hybridization to a cloned human TCGF cDNA probe. CsA completely inhibited induced TCGF mRNA accumulation at concentrations of 0.3-1.0 microgram/ml, whereas low levels of appropriately sized TCGF mRNA were present at 0.01 microgram/ml. In nuclear transcription experiments, CsA inhibited the synthesis of TCGF transcripts in a dose-dependent manner with complete inhibition at a concentration of 1 microgram/ml. In contrast, CsA did not inhibit the expression of two other inducible genes, TCGF receptor and HT-3. Further, HLA gene expression was also less affected than TCGF in CsA-treated cells. These data suggest a relatively selective action of CsA on TCGF gene transcription. Images PMID:6332315

Krönke, M; Leonard, W J; Depper, J M; Arya, S K; Wong-Staal, F; Gallo, R C; Waldmann, T A; Greene, W C

1984-01-01

206

Systems perspectives on mRNA processing  

Microsoft Academic Search

The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers

Adrienne E McKee; Pamela A Silver

2007-01-01

207

mRNA export: threading the needle  

PubMed Central

After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

Gaouar, Ouassila; Germain, Hugo

2013-01-01

208

Self-amplifying mRNA vaccines.  

PubMed

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. PMID:25620012

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

209

Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide  

SciTech Connect

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-{kappa}B DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.

Kaplan, Barbara L.F. [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Ouyang Yanli [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Herring, Amy [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Yea, Sung Su [Department of Biochemistry, College of Medicine, Inje University, Pusan 614-735 (Korea, Republic of); Razdan, Raj [Organix Inc., Woburn, MA 01801 (United States); Kaminski, Norbert E. [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)]. E-mail: kamins11@msu.edu

2005-06-01

210

Two unique mutations in the interleukin-2 receptor gamma chain gene (IL2RG) cause X-linked severe combined immunodeficiency arising in opposite parental germ lines  

SciTech Connect

The gene encoding the gamma chain of the lymphocyte receptor for IL-2 lies in human X13.1 and is mutated in males with X-linked severe combined immunodeficiency (SCID). 27 X-linked SCID mutations have been found in our laboratory. Single strand conformation polymorphism (SSCP) analysis of genomic DNA using primers flanking each of the 8 exons was followed by direct sequencing of abnormally migrating fragments from SCID patients and family members. A 9 bp in-frame duplication insertion was found in IL2RG exon 5 of a patient from a large X-linked SCID pedigree; the resulting duplication of 3 extracellular amino acids, including the first tryptophan of the {open_quotes}WSXWS{close_quotes} cytokine binding motif, is predicted to disrupt interaction of the cytokine receptor chain with its ligand. Genetic linkage studies demonstrated that the grandmaternal X chromosome associated with SCID was contributed to 3 daughters, 2 obligate carriers and 1 woman of unknown status. However, this grandmother`s genomic DNA did not contain the insertion mutation, nor did she have skewed X-chromosome inactivation in her lymphocytes. That both obligate carrier daughters, but not the third daughter, had the insertion proved the grandmother to be a germline mosaic. A second proband had X-linked SCID with a branch point mutation due to substitution of T for A 15 bp 5{prime} of the start of IL2RG exon 3. This mutation resulted in undetectable IL2RG mRNA by Northern blot. Linkage analysis and sequencing of IL2RG DNA in this family proved the mutation to have originated in the germline of the proband`s grandfather, an immunocompetent individual who contributed an X chromosome with normal IL2RG to one daughter and a mutated X to the another.

Puck, J.M.; Pepper, A.E. [National Institutes of Health, Bethesda, MD (United States)

1994-09-01

211

mRNA stability in the nucleus*  

PubMed Central

Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases (RNases). The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase II in eukaryotes. PMID:24793762

Liu, Han; Luo, Min; Wen, Ji-kai

2014-01-01

212

Sensitivity of mRNA Translation  

E-print Network

Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, exit, and elongation rates along the mRNA strand on the steady state protein translation rate. We focus on two special cases where exact closed-form expressions for the translation rate sensitivity can be derived. We discuss the ramifications of our results in the context of functional genomics, molecular evolution, and synthetic biology.

Gilad Poker; Michael Margaliot; Tamir Tuller

2014-09-18

213

Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime-DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin-2-producing CD4 T-cell frequency post-vaccination.  

PubMed

The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. Simple measurement of interferon-? frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T-cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette-Guérin (BCG) prime-Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG-vaccinated mice, and the reduction in colony-forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin-2 (IL-2) -producing CD4 T cells and increased IL-2 production when measured as integrated mean fluorescence intensity post-vaccination as well. These data suggest that measurement of elevated frequency of IL-2-producing CD4 T cells or IL-2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime-boost strategies may be a useful approach to the control of M. tuberculosis infection. PMID:24965530

Kang, Han; Yuan, Qin; Ma, Hui; Hu, Zhi-Dong; Han, De-Ping; Wu, Kang; Lowrie, Douglas B; Fan, Xiao-Yong

2014-12-01

214

Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins.  

PubMed

Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigen-loaded dendritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. In the present study, monocyte-derived DCs from nontreated HIV-1-seropositive patients were electroporated with codon-optimized ("humanized") mRNA encoding consensus HxB-2 (hHXB-2) Gag protein. These DCs elicited a strong HIV-1 Gag-specific interferon-gamma (IFN-gamma) response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DCs also triggered IFN-gamma secretion by autologous peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD8+ T cells from all patients tested. Next, a novel strategy was developed using autologous virus sequences. Significant specific IFN-gamma T-cell responses were induced in all patients tested by DCs electroporated with patients' autologous polymerase chain reaction (PCR)-amplified and in vitro-transcribed proviral and plasma viral mRNA encoding either Gag or Env. The stimulatory effect was seen on PBMCs, CD8+ T cells, and CD4+ T cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin-2 (IL-2) T-cell response was induced by DCs electroporated with hHxB-2 or proviral gag mRNA. These findings open a major perspective for the development of patient-specific immunotherapy for HIV-1 disease. PMID:16263796

Van Gulck, Ellen R A; Ponsaerts, Peter; Heyndrickx, Leo; Vereecken, Katleen; Moerman, Filip; De Roo, Ann; Colebunders, Robert; Van den Bosch, Glenn; Van Bockstaele, Dirk R; Van Tendeloo, Viggo F I; Allard, Sabine; Verrier, Bernard; Marañón, Concepción; Hoeffel, Guillaume; Hosmalin, Anne; Berneman, Zwi N; Vanham, Guido

2006-03-01

215

Inhibition of protein synthesis stabilizes histone mRNA  

SciTech Connect

The inhibition of protein synthesis in exponentially growing S49 cells leads to a specific fivefold increase in histone mRNA in 30 min. The rate of transcription of histone mRNA, measured in intact or digitonin-permeabilized cells, is increased slightly, if at all, by cycloheximide inhibition of protein synthesis. Both approach-to-equilibrium labeling and pulse-chase experiments show that cycloheximide prolongs histone mRNA half-life from approximately 30 min to > 2 h. Histone mRNA made before the addition of cycloheximide becomes stable after the inhibition of protein synthesis, whereas removal of the inhibitor is followed by rapid degradation of histone mRNA. This suggests that the increased stability of histone mRNA during inhibition of protein synthesis results not from alteration of the structure of the mRNA, but from the loss of an activity in the cell which regulates histone mRNA turnover.

Stimac, E.; Groppi, V.E. Jr.; Coffino, P.

1984-10-01

216

Regulation of yeast development by mRNA methylation  

E-print Network

The internal methylation of mRNA post-transcriptionally is an essential component of the mRNA editing machinery in virtually every eukaryotic system. Despite this ubiquity, little is known about the relevance, consequences ...

Agarwala, Sudeep D

2012-01-01

217

Understanding regulation of mRNA by RNA binding proteins  

E-print Network

Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA ...

Robertson, Alexander De Jong

2014-01-01

218

Conventional and unconventional mechanisms for capping viral mRNA  

Microsoft Academic Search

In the eukaryotic cell, capping of mRNA 5? ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5?–3? exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5? ends with either a

Etienne Decroly; François Ferron; Julien Lescar; Bruno Canard

2011-01-01

219

Early Phosphatidylinositol 3Kinase\\/Akt Pathway Activation Limits Poliovirus-Induced JNK-Mediated Cell Death  

Microsoft Academic Search

Poliovirus (PV)-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dys- function mediated by early JNK activation in IMR5 neuroblastoma cells. We showed here that PV simulta- neously activates the phosphatidylinositol 3-kinase (PI3K)\\/Akt survival signaling pathway in these cells, limiting the

Arnaud Autret; Sandra Martin-Latil; Cynthia Brisac; Laurence Mousson; Florence Colbere-Garapin; Bruno Blondel

2008-01-01

220

Early phosphatidylinositol 3-kinase/Akt pathway activation limits1 poliovirus-induced JNK-mediated cell death2  

E-print Network

apoptosis in vitro in tissue cultures of human colon carcinoma cells43 (CaCo-2) (4), promonocytic cells (U937) (29), dendritic cells (41), murine L cells expressing44 CD155 (21, 36), HeLa cells (8, 39

Boyer, Edmond

221

Quinazoline analog HMJ-30 inhibits angiogenesis: involvement of endothelial cell apoptosis through ROS-JNK-mediated death receptor 5 signaling.  

PubMed

The aim of the present study was to explore the effect of 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino) quinazoline (HMJ-30) on the anti-angiogenic properties and apoptosis-related mechanism of human umbilical vein endothelial cells (HUVECs). In this study, HMJ-30 dose- and time-dependently inhibited the viability of HUVECs. We also found that HMJ-30 enhanced disruption of tube-like structures and suppressed cell migration in HUVECs after vascular endothelial growth factor (VEGF) induction. HMJ-30 was also observed to inhibit vessel branching and sprouting in chicken chorioallantoic membrane (CAM). Microsprouting induced by VEGF in the rat aortic ring and blood vessel formation in a mouse Matrigel plug were individually suppressed by HMJ-30. In an in vitro study, HMJ-30 induced the apoptotic death of HUVECs as indicated by DNA fragmentation and promoted reactive oxygen species (ROS) production as determined by flow cytometric assay. In addition, extrinsic caspase signaling (caspase-8 and -3) was activated in the HMJ-30-treated HUVECs and their inhibitors were applied to assess the signal transduction. We investigated the upstream of the death receptor pathway and further observed that the levels of death receptor 5 (DR5) and phosphorylated c-Jun N-terminal kinase (JNK) signals were upregulated in HUVECs following HMJ-30 challenge, which was confirmed by a JNK-specific inhibitor (SP600125). Hence, HMJ-30-induced endothelial cell apoptosis involved the ROS/JNK-regulated DR5 pathway. In summary, HMJ-30 may provide a potential therapeutic effect for the anti-vascular targeting of angiogenesis during cancer treatment. PMID:24919794

Lu, Chi-Cheng; Chen, Hao-Ping; Chiang, Jo-Hua; Jin, Yi-An; Kuo, Sheng-Chu; Wu, Tian-Shung; Hour, Mann-Jen; Yang, Jai-Sing; Chiu, Yu-Jen

2014-08-01

222

ANALYSIS OF CYTOPLASMIC mRNA DECAY IN SACCHAROMYCES CEREVISIAE  

PubMed Central

The yeast, Saccharomyces cerevisiae, is a model system for the study of eukaryotic mRNA degradation. In this organism, a variety of methods have been developed to measure mRNA decay rates, trap intermediates in the mRNA degradation process, and establish precursor–product relationships. In addition, the use of mutant strains lacking specific enzymes involved in mRNA destruction, or key regulatory proteins, allows one to determine the mechanisms by which individual mRNAs are degraded. In this chapter, we discuss methods for analyzing mRNA degradation in S. cerevisiae. PMID:19111187

Passos, Dario O.; Parker, Roy

2010-01-01

223

The Majority of Epidermal T Cells in Psoriasis Vulgaris Lesions can Produce Type 1 Cytokines, Interferon-?, Interleukin2, and Tumor Necrosis Factor-?, Defining TC1 (Cytotoxic T Lymphocyte) and TH1 Effector Populations:1 a Type 1 Differentiation Bias is also Measured in Circulating Blood T Cells in Psoriatic Patients  

Microsoft Academic Search

Psoriasis vulgaris is a skin disease potentially mediated by pro-inflammatory cytokines produced by type 1 lesional T cells. The capability of individual T cells to produce these cytokines in lesional skin is not known. In this study we measured the ability of lesional and peripheral blood T cells to produce intracellular interferon-?, tumor necrosis factor-?, interleukin-2, interleukin-4, and interleukin-10 proteins

Lisa M Austin; Maki Ozawa; Toyoko Kikuchi; Ian B Walters; James G Krueger

1999-01-01

224

Messenger RNA (mRNA) nanoparticle tumour vaccination  

NASA Astrophysics Data System (ADS)

Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

2014-06-01

225

Messenger RNA (mRNA) nanoparticle tumour vaccination.  

PubMed

Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research. PMID:24904987

Phua, Kyle K L; Nair, Smita K; Leong, Kam W

2014-07-21

226

Serum levels of soluble Fas, soluble tumor necrosis factor-receptor II, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus genotype-4  

PubMed Central

Background Liver disease progression from chronic hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) is associated with an imbalance between T-helper 1 and T-helper 2 cytokines. Evaluation of cytokines as possible candidate biomarkers for prediction of HCC was performed using soluble Fas (sFas), soluble tumor necrosis factor receptor-II (sTNFR-II), interleukin-2 receptor (IL-2R) and interleukin-8 (IL-8). Results The following patients were recruited: 79 with HCV infection, 30 with HCC, 32 with chronic liver disease associated with elevated liver enzyme levels (with or without cirrhosis) in addition to 17 with chronic HCV with persistent normal alanine aminotransferase levels (PNALT). Nine normal persons negative either for HCV or for hepatitis B virus were included as a control group. All persons were tested for sFas, sTNFR-II, IL-2R and IL-8 in their serum by quantitative ELISA. HCC patients had higher levels of liver enzymes but lower log-HCV titer when compared to the other groups. HCC patients had also significantly higher levels of sFas, sTNFR-II and IL-2R and significantly lower levels of IL-8 when compared to the other groups. Exclusion of HCC among patients having PNALT could be predicted with 90% sensitivity and 70.6% specificity when sTNFR-II is ? 389 pg/ml or IL-8 is < 290 pg/ml. Conclusions Serum TNFR-II, IL-2R? and IL-8, may be used as combined markers in HCV-infected cases for patients at high risk of developing HCC; further studies, however, are mandatory to check these findings before their application at the population level. PMID:20051112

2010-01-01

227

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.  

PubMed Central

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7777545

Maass, G; Schmidt, W; Berger, M; Schilcher, F; Koszik, F; Schneeberger, A; Stingl, G; Birnstiel, M L; Schweighoffer, T

1995-01-01

228

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-?  

PubMed Central

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-? were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-? (ChIFN-?) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-? did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV. PMID:19461208

Park, Jeong Ho; Sung, Haan Woo; Yoon, Byung Il

2009-01-01

229

Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer  

PubMed Central

Background We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer. Patients and methods Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-?. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered. Results Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT. Conclusion AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer. PMID:24947606

2014-01-01

230

Interleukin 2 inhibitor in synovial fluid.  

PubMed Central

Since evidence for the presence of IL-2 activity in rheumatoid synovial fluid is conflicting, we have assayed IL-2 activity in synovial fluid from patients with rheumatoid arthritis (RA) and other articular diseases (OAD). Using the IL-2-dependent murine T cell line CTLL, IL-2 activity was not demonstrable in synovial fluid tested at concentrations ranging from 50% to 0.02%. There was an inhibitory effect on IL-2 activity in the bioassay of synovial fluid from 16 of the 22 patients with RA and 15 of the 16 with OAD. This inhibitory activity was heat-labile, precipitable by ammonium sulphate, reversible with excess IL-2 and was not significantly altered by preincubation of synovial fluid with CTLL. The mean inhibitory activity of synovial fluid from patients with RA was significantly reduced in comparison with that of synovial fluid from patients with OAD. Sera also had an inhibitory effect on IL-2 activity; however sera from patients with RA were less inhibitory than control sera but were more inhibitory than sera from patients with systemic lupus erythematosus. The deficiency in synovial fluid of an inhibitor of IL-2 activity may be relevant to the pathogenesis of RA. PMID:3260839

Emery, P; Gentry, K C; Kelso, A; Mackay, I R

1988-01-01

231

Interleukin-2 Engineering for improved therapeutic effectiveness  

E-print Network

(cont.) (K[d] [approximately] 10pM) for its private alpha receptor subunit, unlike wild-type IL-2 (K[d] [approximately] 10 nM). IL-2 mutants with picomolar affinity for IL-2R? stimulate T cell growth responses quantitatively ...

Rao, Balaji Madhav, 1978-

2004-01-01

232

Engineering persistent interleukin-2 for cancer immunotherapy  

E-print Network

Mobilizing the immune system to recognize and destroy tumor cells is a promising strategy for treating cancer. In contrast to standard therapeutic approaches such as surgery, radiation, and chemotherapy, immunotherapy ...

Gai, Shuning

2012-01-01

233

Transcriptional Elongation and mRNA Export Are Coregulated Processes  

PubMed Central

Chromatin structure complexity requires the interaction and coordinated work of a multiplicity of factors at different transcriptional regulation stages. Transcription control comprises a set of processes that ensures proper balance in the gene expression under different conditions, such as signals, metabolic states, or development. We could frame those steps from epigenetic marks to mRNA stability to support the holistic view of a fine-tune balance of final mRNA levels through mRNA transcription, export, stability, translation, and degradation. Transport of mRNA from the nucleus to the cytoplasm is a key process in regulated gene expression. Transcriptional elongation and mRNA export are coregulated steps that determine the mature mRNA levels in the cytoplasm. In this paper, recent insights into the coordination of these processes in eukaryotes will be summarised. PMID:22567364

Molina-Navarro, Maria Micaela; Martinez-Jimenez, Celia Pilar; Rodriguez-Navarro, Susana

2011-01-01

234

Highly Efficient Expression of Interleukin-2 under the Control of Rabbit ?-Globin Intron II Gene Enhances Protective Immune Responses of Porcine Reproductive and Respiratory Syndrome (PRRS) DNA Vaccine in Pigs  

PubMed Central

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit ?-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections. PMID:24603502

Liu, Jiyu; Hu, Yue; Li, Feng; Wu, Jiaqiang; Guo, Lihui; Liu, Junzhen; Tao, Haiying; Sun, Wenbo; Chen, Lei; Cong, Xiaoyan; Ren, Sufang; Shi, Jianli; Li, Jun; Wang, Jinbao; Huang, Baohua; Wan, Renzhong

2014-01-01

235

Adjuvant low-dose interleukin-2 (IL-2) plus interferon-? (IFN-?) in operable renal cell carcinoma (RCC): a phase III, randomized, multicentre trial of the Italian Oncology Group for Clinical Research (GOIRC).  

PubMed

There is currently no standard therapy to reduce the recurrence rate after surgery for renal cell carcinoma (RCC). The aim of this study was to assess efficacy and safety of adjuvant treatment with low doses of interleukin-2 (IL-2)+interferon-? (IFN-?) in operable RCC. The patients were randomized 1:1 to receive a 4-week cycle of low-dose IL-2+IFN-? or observation after primary surgery for RCC. Treatment cycles were repeated every 4 months for the first 2 years and every 6 months for the subsequent 3 years. The primary endpoint was recurrence-free survival (RFS); safety; and overall survival (OS) were secondary endpoints. ClinicalTrials.gov registration number was NCT00502034. 303/310 randomized patients (156 in the immunotherapy arm and 154 in the observation group) were evaluable at the intention-to-treat analyses. The 2 arms were well balanced. At a median follow-up of 52 months (range, 12-151 mo), RFS, and OS were similar, with an estimated hazard ratio (HR) of 0.84 [95% confidence interval (CI), 0.54-1.31; P=0.44] and of 1.07 (95% CI, 0.64-1.79; P=0.79), respectively in the 2 groups. Unplanned, subgroup analysis showed a positive effect of the treatment for patients with age 60 years and younger, pN0, tumor grades 1-2, and pT3a stage. Among patients with the combined presence of ? 2 of these factors, immunotherapy had a positive effect on RFS (HR=0.44; 95% CI, 0.24-0.82; P ? 0.01), whereas patients with <2 factors in the treatment arm exhibited a significant poorer OS (HR=2.27; 95% CI, 1.03-5.03 P=0.037). Toxicity of immunotherapy was mild and limited to World Health Organization grade 1-2 in most cases. Adjuvant immunotherapy with IL-2+IFN-? showed no RFS or OS improvement in RCC patients who underwent radical surgery. The results of subset analysis here presented are only hypothesis generating. PMID:25304727

Passalacqua, Rodolfo; Caminiti, Caterina; Buti, Sebastiano; Porta, Camillo; Camisa, Roberta; Braglia, Luca; Tomasello, Gianluca; Vaglio, Augusto; Labianca, Roberto; Rondini, Ermanno; Sabbatini, Roberto; Nastasi, Giuseppe; Artioli, Fabrizio; Prati, Andrea; Potenzoni, Michele; Pezzuolo, Debora; Oliva, Elena; Alberici, Federico; Buzio, Carlo

2014-01-01

236

MELEAGRIS GALLOPAVO PROGLUCAGON (GCG), CLASS A MRNA, COMPLETE CDS  

Technology Transfer Automated Retrieval System (TEKTRAN)

In mammals, the proglucagon gene produces a single identical mRNA transcript in pancreas and intestine. This transcript encodes glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2). Previously, we identified two distinct proglucagon mRNA transcripts in chickens, one that encodes glucag...

237

MELEAGRIS GALLOPAVO PROGLUCAGON (GCG), CLASS B MRNA, COMPLETE CDS  

Technology Transfer Automated Retrieval System (TEKTRAN)

In mammals, the proglucagon gene produces a single identical mRNA transcript in pancreas and intestine. This transcript encodes glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2). Previously, we identified two distinct proglucagon mRNA transcripts in chickens, one that encodes glucag...

238

Tri- to be mono- for bacterial mRNA decay.  

PubMed

Messing et al. (2009) report the homodimeric structure of the Bdellovibrio bacteriovorus RppH pyrophosphohydrolase, which hydrolyzes the mRNA 5' triphosphate to initiate bacterial mRNA decay. These structures reveal insights into BdRppH substrate recognition and analogies to eukaryotic decapping enzymes. PMID:19278643

Bail, Sophie; Kiledjian, Megerditch

2009-03-11

239

ORIGINAL PAPER Quantifying mRNA levels across tissue sections  

E-print Network

ORIGINAL PAPER Quantifying mRNA levels across tissue sections with 2D-RT-qPCR Michael Armani & Michael A. Tangrea & Benjamin Shapiro & Michael R. Emmert-Buck & Elisabeth Smela Received: 16 March 2011 Abstract Measurement of mRNA levels across tissue sam- ples facilitates an understanding of how genes

Shapiro, Benjamin

240

Co-translational mRNA decay in Saccharomyces cerevisiae  

PubMed Central

The rates of RNA decay and transcription determine the steady state levels of all mRNAs and both can be subject to regulation. While the details of transcriptional regulation are becoming increasingly understood, the mechanism(s) controlling mRNA decay remain unclear. In yeast, a major pathway of mRNA decay begins with deadenylation followed by decapping and 5’-3’ exonuclease digestion. Importantly, it is hypothesized that ribosomes must be removed from mRNA before transcripts are destroyed. Contrary to this prediction, here we show that decay takes place while mRNAs are associated with actively translating ribosomes. The data indicate that dissociation of ribosomes from mRNA is not a prerequisite for decay and we suggest that the 5’-3’ polarity of mRNA degradation has evolved to ensure that the last translocating ribosome can complete translation. PMID:19701183

Hu, Wenqian; Sweet, Thomas J.; Chamnongpol, Sangpen; Baker, Kristian E.; Coller, Jeff

2009-01-01

241

CELFish ways to modulate mRNA decay  

PubMed Central

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

242

Selective Localization of Arc mRNA in Dendrites Involves Activity- and Translation-Dependent mRNA Degradation  

PubMed Central

Arc is an immediate early gene that is unique among neuronal mRNAs because its transcripts are transported into dendrites and accumulate near activated synapses, presumably to be translated locally. These qualities pose Arc as playing an important, yet not fully understood, role in the activity-dependent modifications of synapses that are thought to underlie memory storage. Here we show in vivo in rats that newly synthesized Arc mRNA accumulates at activated synapses and that synaptic activity simultaneously triggers mRNA decay that eliminates Arc mRNA from inactive dendritic domains. Arc mRNA degradation occurs throughout the dendrite and requires both NMDA receptor activation and active translation. Synaptic activation did not lead to decreases in another dendritic mRNA (?CaMKII), indicating that there is not a general activation of mRNA degradation in dendrites. These data reveal a novel mechanism for controlling mRNA distribution within dendrites and highlight activity-dependent mRNA degradation as a regulatory process involved in synaptic plasticity. PMID:24671994

Farris, Shannon; Lewandowski, Gail; Cox, Conor D.

2014-01-01

243

Quality Control of Bacterial mRNA Decoding and Decay  

PubMed Central

Studies in eukaryotes and prokaryotes have revealed that gene expression is not only controlled through altering the rate of transcription but also through varying rates of translation and mRNA decay. Indeed, the expression level of a protein is strongly affected by the steady state level of its mRNA. RNA decay can, along with transcription, play an important role in regulating gene expression by fine-tuning the steady state level of a given transcript and affecting its subsequent decoding during translation. Alterations in mRNA stability can in turn have dramatic effects on cell physiology and as a consequence the fitness and survival of the organism. Recent evidence suggests that mRNA decay can be regulated in response to environmental cues in order to enable the organism to adapt to its changing surroundings. Bacteria have evolved unique post transcriptional control mechanisms to enact such adaptive responses through: 1) general mRNA decay, 2) differential mRNA degradation using small non-coding RNAs (sRNAs), and 3) selective mRNA degradation using the tmRNA quality control system. Here, we review our current understanding of these molecular mechanisms, gleaned primarily from studies of the model gram negative organism E. coli, that regulate the stability and degradation of normal and defective transcripts. PMID:18342642

Richards, Jamie; Sundermeier, Thomas; Svetlanov, Anton; Karzai, A. Wali

2008-01-01

244

Mechanism of grk mRNA anchoring during Drosophila oogenesis   

E-print Network

Messenger RNA localization is a widespread mechanism of posttranscriptional regulation of gene expression in multicellular organisms ranging from yeast to mammals. In Drosophila oocytes, gurken (grk) mRNA is transported ...

Soetaert, Jan

2009-01-01

245

Polyadenylation of Vesicular Stomatitis Virus mRNA  

PubMed Central

Vesicular stomatitis virus (VSV) mRNA isolated from infected cell polysomes contains polyadenylic acid [poly(A)] sequences. Detergent-activated purified virions in vitro can transcribe complementary RNA, which has sedimentation properties similar to mRNA, and this RNA also contains poly(A) sequences. Digestion of virion RNA with U2 RNase under conditions where hydrolysis is specific for purine linkages leaves no sequences of polyuridylic acid corresponding in length to the poly(A) on the transcripts. Growth of infectious virus is not inhibited by 3-deoxyadenosine (cordycepin) under conditions in which it inhibits polyadenylation of cellular mRNA. The virus-specific mRNA produced in the presence of cordycepin has poly(A) sequences of the same size distribution as that synthesized in the absence of cordycepin. PMID:4363251

Ehrenfeld, Ellie

1974-01-01

246

Structure and stability correlation of an mRNA pseudoknot  

E-print Network

heptanucleotide sequence and a downstream pseudoknot are essential for mRNA frameshifting. The BWYV pseudoknot is a classical H-type pseudoknot containing two helical stems and two connecting loop regions. The loop-stem interactions have been proposed...

Suram, Saritha

2002-01-01

247

Regulation of mRNA Translation and Stability  

E-print Network

, plants, and protozoa. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3 regulators of gene expression in metazoan animals, plants, and protozoa. In mammals, miRNAs are predicted

Bedwell, David M.

248

Le interferon mRNA from human fibroblasts.  

PubMed Central

Human F and Le interferon can be clearly distinguished on the basis of different antigenic properties and host range. After inoculation with Newcastle disease virus (NDV), GM-258 fibroblasts produced Le as well as F interferon; in contrast, only F interferon was detectable after stimulation with poly(I) . poly(C). Polyadenylylated mRNA isolated from fibroblasts induced with poly(I) . poly(C) or NDV was injected into Xenopus laevis oocytes and the interferon activities thus produced were analyzed. Only F interferon production was demonstrable in oocytes injected with mRNA from cells induced with poly(I) . poly(C), whereas both F and Le interferons were made in oocytes injected with mRNA from NDV-induced cultures. The time course of accumulation of F and Le interferon mRNAs in NDV-induced cells corresponded to the kinetics of F and Le interferon synthesis in intact cells. The ratio of F and Le interferons made in oocytes was similar to that observed in intact GM-258 cells. F and Le interferon mRNA activities isolated from GM-258 cells could not be separated by sucrose density gradient centrifugation. However, the profile of F mRNA activity was more heterogeneous and its peak sedimented somewhat more slowly than that of Le interferon mRNA. These results suggest that the varying ratios of F and Le interferon synthesis in different cells after different modes of stimulation are determined at the level of mRNA. The induction mechanisms of F and Le interferon mRNA synthesis appear to be closely related but not identical. PMID:6159644

Pang, R H; Hayes, T G; Vilcek, J

1980-01-01

249

Expression of TREM-1 mRNA in acute pancreatitis  

Microsoft Academic Search

AIM: To explore the expression of triggering receptor expressed on myeloid cells (TREM-1) mRNA in acute pancreatitis (AP). METHODS: Using the reverse transcription polymerase chain reaction (RT-PCR), we examined the expression of TREM-1 mRNA in 10 cases of mild acute pancreatitis (MAP), 8 cases of severe acute pancreatitis (SAP), and 10 cases of healthy control subjects. And we also examined

Da-Yu Wang; Ren-Yi Qin; Zheng-Ren Liu; Manoj Kumar Gupta; Qing Chang; Manoj Kumar

250

Mechanisms of endonuclease-mediated mRNA decay  

PubMed Central

Endonuclease cleavage was one of the first identified mechanisms of mRNA decay but until recently it was thought to play a minor role to the better-known processes of deadenylation, decapping and exonuclease-catalyzed decay. Most of the early examples of endonuclease decay came from studies of a particular mRNA whose turnover changed in response to hormone, cytokine, developmental or nutritional stimuli. Only a few of these examples of endonuclease-mediated mRNA decay progressed to the point where the enzyme responsible for the initiating event was identified and studied in any detail. The discovery of microRNAs and RISC-catalyzed endonuclease cleavage followed by the identification of PIN (pilT N-terminal) domains that impart endonuclease activity to a number of the proteins involved in mRNA decay has led to a resurgence of interest in endonuclease-mediated mRNA decay. PIN domains show no substrate selectivity, and their involvement in a number of decay pathways highlights a recurring theme that the context in which an endonuclease functions is a primary factor in determining whether any given mRNA will be targeted for decay by this or the default exonuclease-mediated decay processes. PMID:21957046

Schoenberg, Daniel R.

2012-01-01

251

HDAC3 regulates stability of estrogen receptor ? mRNA  

SciTech Connect

Highlights: ? HDAC inhibitors decrease the stability of ER? mRNA in MCF-7 cells. ? HDAC3 is involved in maintaining ER? mRNA stability in MCF-7 cells. ? ER? mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ER?-positive MCF-7 cells. ? HDAC3 specific inhibitor will be one of new drugs for ER?-positive breast cancers. -- Abstract: Estrogen receptor alpha (ER?) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ER? expression in ER?-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ER? mRNA, and that knockdown of HDAC3 decreases the stability of ER? mRNA and suppresses estrogen-dependent proliferation of ER?-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ER?-positive tumors are higher than those in ER?-negative tumors, thus suggesting that HDAC3 is necessary for ER? mRNA stability, and is involved in the estrogen-dependent proliferation of ER?-positive tumors.

Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail: junny@agbi.tsukuba.ac.jp

2013-03-08

252

Interplay between viruses and host mRNA degradation  

PubMed Central

Messenger RNA degradation is a fundamental cellular process that plays a critical role in regulating gene expression by controlling both the quality and the abundance of mRNAs in cells. Naturally, viruses must successfully interface with the robust cellular RNA degradation machinery to achieve an optimal balance between viral and cellular gene expression and establish a productive infection in the host. In the past several years, studies have discovered many elegant strategies that viruses have evolved to circumvent the cellular RNA degradation machinery, ranging from disarming the RNA decay pathways and co-opting the factors governing cellular mRNA stability to promoting host mRNA degradation that facilitates selective viral gene expression and alters the dynamics of host-pathogen interaction. This review summarizes the current knowledge of the multifaceted interaction between viruses and cellular mRNA degradation machinery to provide an insight into the regulatory mechanisms that influence gene expression in viral infections. PMID:23274304

Narayanan, Krishna; Makino, Shinji

2013-01-01

253

mRNA Degradation Machinery in Plants Yukako Chiba & Pamela J. Green  

E-print Network

REVIEW mRNA Degradation Machinery in Plants Yukako Chiba & Pamela J. Green Received: 23 February, much less information is available regarding mRNA degradation machineries. How- ever, in the past of machineries involved in general mRNA degradation and mRNA surveillance systems in plants. Introduction

Green, Pamela

254

Regulation of mRNA Trafficking by Nuclear Pore Complexes  

PubMed Central

Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

Bonnet, Amandine; Palancade, Benoit

2014-01-01

255

Multiple mRNA Decapping Enzymes in Mammalian Cells  

PubMed Central

SUMMARY Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5?-end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover. PMID:21070968

Song, Man-Gen; Li, You; Kiledjian, Megerditch

2010-01-01

256

BIOMARKERS OF ENDOCRINE DISRUPTION AT THE MRNA LEVEL  

EPA Science Inventory

Denslow, Nancy D., Christopher J. Bowman, Gillian Robinson, H. Stephen Lee, Ronald J. Ferguson, Michael J. Hemmer and Leroy C. Folmar. 1999. Biomarkers of Endocrine Disruption at the mRNA Level. In: Environmental Toxicology and Risk Assessment: Standardization of Biomarkers for ...

257

Analysis of mRNA recognition by human thymidylate synthase  

PubMed Central

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2?-deoxyuridine-5?-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site. PMID:25423174

Brunn, Nicholas D.; Dibrov, Sergey M.; Kao, Melody B.; Ghassemian, Majid; Hermann, Thomas

2014-01-01

258

Differential protein occupancy profiling of the mRNA transcriptome  

PubMed Central

Background RNA-binding proteins (RBPs) mediate mRNA biogenesis, translation and decay. We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted cis-regulatory mRNA regions without a priori knowledge of the regulatory protein component. Results We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3? UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells. Conclusions We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing cis-regulatory RNA sequence space and its dynamics in growth, development and disease. PMID:24417896

2014-01-01

259

Relationship of ORF length and mRNA degradation in Escherichia coli genome  

NASA Astrophysics Data System (ADS)

The decay rate of mRNA varied in a wide range within a species and the special sequence features were considered as important factor determining this variation. In order to investigate the effect of ORF length on mRNA degradation in Escherichia coli genome, the correlation between ORF length and mRNA half-life was calculated in different cultures for mRNA growing. Although the effect of ORF length on mRNA degradation is strongly dependent of the growth medium, a significant negative correlation between ORF length and mRNA half-life is observed in the mRNAs less affected by the growth medium. In particular, some important genes, such as essential genes, show significant inverse correlation between ORF length and mRNA degradation. Our results indicate that ORF length is an important factor influencing mRNA degradation and the increasing length tends to decrease the mRNA longevity.

Jia, Mengwen; Zhan, Yong

2012-09-01

260

Peptide inhibitors of botulinum neurotoxin by mRNA display  

SciTech Connect

Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

Yiadom, Kwabena P.A.B. [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States); Muhie, Seid [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States); Yang, David C.H. [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States)]. E-mail: yangdc@georgetown.edu

2005-10-07

261

Translation with frameshifting of ribosome along mRNA transcript  

E-print Network

Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

Li, Jingwei

2015-01-01

262

Evaluation of Glycine max mRNA clusters  

PubMed Central

Background Clustering the ESTs from a large dataset representing a single species is a convenient starting point for a number of investigations into gene discovery, genome evolution, expression patterns, and alternatively spliced transcripts. Several methods have been developed to accomplish this, the most widely available being UniGene, a public domain collection of gene-oriented clusters for over 45 different species created and maintained by NCBI. The goal is for each cluster to represent a unique gene, but currently it is not known how closely the overall results represent that reality. UniGene's build procedure begins with initial mRNA clusters before joining ESTs. UniGene's results for soybean indicate a significant amount of redundancy among some sequences reported to be unique mRNAs. To establish a valid non-redundant known gene set for Glycine max we applied our algorithm to the clustering of only mRNA sequences. The mRNA dataset was run through the algorithm using two different matching stringencies. The resulting cluster compositions were compared to each other and to UniGene. Clusters exhibiting differences among the three methods were analyzed by 1) nucleotide and amino acid alignment and 2) submitting authors conclusions to determine whether members of a single cluster represented the same gene or not. Results Of the 12 clusters that were examined closely most contained examples of sequences that did not belong in the same cluster. However, neither the two stringencies of PECT nor UniGene had a significantly greater record of accuracy in placing paralogs into separate clusters. Conclusion Our results reveal that, although each method produces some errors, using multiple stringencies for matching or a sequential hierarchical method of increasing stringencies can provide more reliable results and therefore allow greater confidence in the vast majority of clusters that contain only ESTs and no mRNA sequences. PMID:16026604

Frank, Ronald L; Ercal, Fikret

2005-01-01

263

CELL BIOLOGY: TAPping into mRNA Export  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. There seem to be numerous pathways for exporting mRNAs from the nucleus to the cytoplasm. But working out which set of export adaptors and receptors transport individual mRNAs has been very difficult. In a Perspective, Moore and Rosbash discuss a new strategy using cell-penetrating peptide inhibitors for unraveling the routes of mRNA export in living cells (Gallouzi and Steitz).

Melissa J. Moore (Brandeis University; Department of Biochemistry)

2001-11-30

264

Vibrational force alters mRNA expression in osteoblasts  

NASA Technical Reports Server (NTRS)

Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

1997-01-01

265

Compilation of E. coli mRNA promoter sequences.  

PubMed Central

An updated compilation of 300 E. coli mRNA promoter sequences is presented. For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally. We comment on the reliability of the sequence databanks and analyze the conservation of known promoter features in the current compilation. This database is available by E-mail. PMID:8479900

Lisser, S; Margalit, H

1993-01-01

266

Nuclear mRNA export: insights from virology  

Microsoft Academic Search

To maximize the production of progeny virions, several viruses have evolved mechanisms that promote the selective nuclear export of viral mRNA transcripts while, in some cases, inhibiting the export of cellular mRNAs. To achieve this goal, viruses have evolved regulatory proteins and cis-acting RNA elements that selectively interact with key cellular nuclear export factors. Efforts to identify the cellular targets

Bryan R Cullen

2003-01-01

267

Mechanisms Coordinating ELAV/Hu mRNA Regulons  

PubMed Central

The 5’ and 3’ untranslated regions (UTRs) of messenger RNAs (mRNAs) function as platforms that can determine the fate of each mRNA individually and in aggregate. Multiple mRNAs that encode proteins that are functionally related often interact with RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) that coordinate their expression in time and space as RNA regulons within the ribonucleoprotein (RNP) infrastructure we term the ribonome. Recent ribonomic methods have emerged that can determine which mRNAs are bound and regulated by RBPs and ncRNAs, some of which act in combination to determine global outcomes. ELAV/Hu proteins bind to AU-rich elements (ARE) in mRNAs and regulate their stability from splicing to translation, and the ubiquitous HuR protein has been implicated in cancerous cell growth. Recent work is focused on mechanistic models of how ELAV/Hu proteins increase mRNA stability and translation by repressing microRNAs (miRs) and the RNA induced silencing complex (RISC) via ARE-based ribonucleosomes that may affect global functions of mRNA regulons. PMID:23312841

Simone, Laura E.; Keene, Jack D.

2013-01-01

268

Identification of phloem-mobile mRNA.  

PubMed

Signaling between cells, tissues and organs is essential for multicellular organisms to coordinate and adapt their development and growth to internal and environmental changes. Plants have evolved a plant-specific symplasmic pathway, called plasmodesmata, for efficient intercellular communication, in addition to the receptor-ligand-based apoplasmic pathway. Long-distance signaling between distant organs is enabled via the phloem tube system, where plasmodesmata contribute to phloem loading and unloading for photosynthate allocation. In addition to signaling by small molecules such as metabolites and phytohormones, the transport of proteins, small RNAs and mRNAs is also considered an important mechanism to achieve long-distance signaling in plants. Recent studies on phloem-mobile proteins and small RNAs have revealed their role in crucial physiological processes including flowering, systemic silencing and nutrient allocation. However, the biological role of mRNAs found in the phloem tube is not yet clear, though their mobility over long-distances has been well evidenced. To gain this knowledge, it is important to collect further information on mRNA profiles in the phloem translocation stream. In this review, I summarize the current approaches to identifying the mRNA population in the phloem translocation system, and discuss the possible role of short- and long-distance mRNA transport. PMID:25516498

Notaguchi, Michitaka

2015-01-01

269

Sequence and regulation of European eel prolactin mRNA.  

PubMed

cDNA clones encoding the European eel (Anguilla anguilla L.) prolactin were isolated from a pituitary cDNA library constructed in gamma gt10, using a rainbow trout Prl cDNA fragment as a probe. Four different inserts were subcloned into the pGEM 3Z plasmid after PCR amplification. The 1082 bp-long nucleotide sequence revealed an open reading frame of 627 bp encoding a 24 amino acid-long signal peptide followed by a 185 amino acid-long mature protein. Comparison studies showed 60-70% homology with other known teleost fish prolactins and 30-45% with non-teleost fish, amphibian, reptilian, avian and mammalian prolactins. In situ hybridization studies using labelled prolactin RNA probe showed a strong signal in the rostral pars distalis of the pituitary gland. We next examined the physiological regulation of this prolactin synthesis in vivo using Northern blot analysis and prolactin cDNA probe labelled by random priming. The pituitary prolactin mRNA level was markedly decreased 3 weeks after transfer of eels from freshwater to sea water. Implants of thyroid hormones left for up to three weeks were ineffective on prolactin mRNA. Estradiol administered as implant, alone or in combination with 500 micrograms testosterone, was also unable to significantly alter the pituitary mRNA level for prolactin in the freshwater silver eels whatever the dose used (20-500 micrograms) and whatever the duration of treatment (from 4 days to 10 weeks). PMID:7926267

Quérat, B; Cardinaud, B; Hardy, A; Vidal, B; D'Angelo, G

1994-06-01

270

Regulation of CTR2 mRNA by the nonsense-mediated mRNA decay pathway.  

PubMed

The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3'-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3'-UTRs and are sensitive to NMD, although the extent by which these long 3'-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3'-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3'-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3'-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD. PMID:25257758

Peccarelli, Megan; Scott, Taylor D; Wong, Hoifung; Wang, Xuya; Kebaara, Bessie W

2014-11-01

271

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes  

Microsoft Academic Search

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK

Calivarathan Latchoumycandane; Quee Ming Seah; Rachel C. H. Tan; Jetsumon Sattabongkot; Walter Beerheide; Urs A.. Boelsterli

2006-01-01

272

Arjunolic acid, a triterpenoid saponin, prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection  

Microsoft Academic Search

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug and is safe at therapeutic doses but its overdose frequently causes liver injury. In earlier studies, we demonstrated that arjunolic acid (AA) has a protective effect against chemically induced hepatotoxicity. The purpose of this study was to explore whether AA plays any protective role against APAP-induced acute hepatotoxicity and, if

Jyotirmoy Ghosh; Joydeep Das; Prasenjit Manna; Parames C. Sil

2010-01-01

273

Conceptual Modeling of mRNA Decay Provokes New Hypotheses  

PubMed Central

Biologists are required to integrate large amounts of data to construct a working model of the system under investigation. This model is often informal and stored mentally or textually, making it prone to contain undetected inconsistencies, inaccuracies, or even contradictions, not much less than a representation in free natural language. Using Object-Process Methodology (OPM), a formal yet visual and humanly accessible conceptual modeling language, we have created an executable working model of the mRNA decay process in Saccharomyces cerevisiae, as well as the import of its components to the nucleus following mRNA decay. We show how our model, which incorporates knowledge from 43 articles, can reproduce outcomes that match the experimental findings, evaluate hypotheses, and predict new possible outcomes. Moreover, we were able to analyze the effects of the mRNA decay model perturbations related to gene and interaction deletions, and predict the nuclear import of certain decay factors, which we then verified experimentally. In particular, we verified experimentally the hypothesis that Rpb4p, Lsm1p, and Pan2p remain bound to the RNA 3?-untralslated region during the entire process of the 5? to 3? degradation of the RNA open reading frame. The model has also highlighted erroneous hypotheses that indeed were not in line with the experimental outcomes. Beyond the scientific value of these specific findings, this work demonstrates the value of the conceptual model as an in silico vehicle for hypotheses generation and testing, which can reinforce, and often even replace, risky, costlier wet lab experiments. PMID:25255440

Somekh, Judith; Haimovich, Gal; Guterman, Adi; Dori, Dov; Choder, Mordechai

2014-01-01

274

Cyclosporin A Inhibits T-Cell Growth Factor Gene Expression at the Level of mRNA Transcription  

Microsoft Academic Search

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function.

Martin Kronke; Warren J. Leonard; Joel M. Depper; Suresh K. Arya; Flossie Wong-Staal; Robert C. Gallo; Thomas A. Waldmann; Warner C. Greene

1984-01-01

275

Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo  

E-print Network

frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular m

Ares Jr., Manny

276

Nonstop mRNA Decay Initiates at the Ribosome  

PubMed Central

SUMMARY The translation machinery deciphers genetic information encoded within mRNAs to synthesize proteins needed for various cellular functions. Defective mRNAs that lack in-frame stop codons trigger nonproductive stalling of ribosomes. We investigated how cells deal with such defective mRNAs, and present evidence to demonstrate that RNase R, a processive 3?-to-5? exoribonuclease, is recruited to stalled ribosomes for the specific task of degrading defective mRNAs. The recruitment process is selective for nonstop mRNAs and is dependent on the activities of SmpB protein and tmRNA. Most intriguingly, our analysis reveals that a unique structural feature of RNase R, the C-terminal lysine-rich (K-rich) domain, is required both for productive ribosome engagement and targeted nonstop mRNA decay activities of the enzyme. These findings provide new insights into how a general RNase is recruited to the translation machinery and highlight a novel role for the ribosome as a platform for initiating nonstop mRNA decay. PMID:21091502

Ge, Zhiyun; Mehta, Preeti; Richards, Jamie; Karzai, A. Wali

2010-01-01

277

Classifying Gene Expression Profiles from Pairwise mRNA Comparisons*  

PubMed Central

We present a new approach to molecular classification based on mRNA comparisons. Our method, referred to as the top-scoring pair(s) (TSP) classifier, is motivated by current technical and practical limitations in using gene expression microarray data for class prediction, for example to detect disease, identify tumors or predict treatment response. Accurate statistical inference from such data is difficult due to the small number of observations, typically tens, relative to the large number of genes, typically thousands. Moreover, conventional methods from machine learning lead to decisions which are usually very difficult to interpret in simple or biologically meaningful terms. In contrast, the TSP classifier provides decision rules which i) involve very few genes and only relative expression values (e.g., comparing the mRNA counts within a single pair of genes); ii) are both accurate and transparent; and iii) provide specific hypotheses for follow-up studies. In particular, the TSP classifier achieves prediction rates with standard cancer data that are as high as those of previous studies which use considerably more genes and complex procedures. Finally, the TSP classifier is parameter-free, thus avoiding the type of over-fitting and inflated estimates of performance that result when all aspects of learning a predictor are not properly cross-validated. PMID:16646797

Geman, Donald; d'Avignon, Christian; Naiman, Daniel Q.; Winslow, Raimond L.

2006-01-01

278

Subcellular mRNA localisation at a glance  

PubMed Central

ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms. PMID:24833669

Parton, Richard M.; Davidson, Alexander; Davis, Ilan; Weil, Timothy T.

2014-01-01

279

Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro  

PubMed Central

Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1? cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1? generated 2?, 3?-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA. PMID:21398633

Shinya, Sayoko; Kadokura, Hiroshi; Imagawa, Yusuke; Inoue, Michihiro; Yanagitani, Kota; Kohno, Kenji

2011-01-01

280

mRNA retroposition in human cells: processed pseudogene formation.  

PubMed Central

Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome. Images PMID:8557053

Maestre, J; Tchénio, T; Dhellin, O; Heidmann, T

1995-01-01

281

Prolyl carboxypeptidase mRNA expression in the mouse brain.  

PubMed

Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and ?-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic ?-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain. PMID:24161824

Jeong, Jin Kwon; Diano, Sabrina

2014-01-13

282

Clustered bottlenecks in mRNA translation and protein synthesis  

E-print Network

We construct an algorithm that generates large, band-diagonal transition matrices for a totally asymmetric exclusion process (TASEP) with local hopping rate inhomogeneities. The matrices are diagonalized numerically to find steady-state currents of TASEPs with local variations in hopping rate. The results are then used to investigate clustering of slow codons along mRNA. Ribosome density profiles near neighboring clusters of slow codons interact, enhancing suppression of ribosome throughput when such bottlenecks are closely spaced. Increasing the slow codon cluster size, beyond $\\approx 3-4$, does not significantly reduce ribosome current. Our results are verified by extensive Monte-Carlo simulations and provide a biologically-motivated explanation for the experimentally-observed clustering of low-usage codons.

Tom Chou; Greg Lakatos

2003-10-29

283

Analyzing Subcellular mRNA Localization via Cell Fractionation  

PubMed Central

Summary The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon. The partitioning of mRNAs to the ER is generally thought to be achieved by the signal recognition particle (SRP) pathway. In this pathway, mRNA localization to the ER is determined by the translation product – translation yields an N-terminal signal sequence or topogenic signal that is recognized by the SRP and the resulting mRNA-ribosome-SRP complex is then recruited to the ER membrane. Recent studies have demonstrated that mRNAs can be localized to the ER via a signal sequence and/or translation-independent pathway(s) and that discrete sets of cytosolic protein-encoding mRNAs are enriched on the ER membrane, though they lack an encoded signal sequence. These key findings reopen investigations into the mechanism(s) that govern mRNA localization to the ER. In this contribution, we describe two independent methods that can be utilized to study this important and poorly understood aspect of eukaryotic cell biology. These methods comprise two independent means of fractionating tissue culture cells to yield free/cytosolic polyribosomes and ER membrane-bound polyribosomes. Detailed methods for the fractionation and characterization of the two polyribosome pools are provided. PMID:21431749

Jagannathan, Sujatha; Nwosu, Christine; Nicchitta, Christopher V.

2013-01-01

284

An agent-based model for mRNA export through the nuclear pore complex  

PubMed Central

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics. PMID:25253717

Azimi, Mohammad; Bulat, Evgeny; Weis, Karsten; Mofrad, Mohammad R. K.

2014-01-01

285

The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.  

PubMed Central

The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

1996-01-01

286

Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C  

PubMed Central

AIM: To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer’s instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile. PMID:25852271

Kasprzak, Aldona; Adamek, Agnieszka; Przybyszewska, Wies?awa; Pyda, Przemys?aw; Szmeja, Jacek; Seraszek-Jaros, Agnieszka; Lanzafame, Agata; Surdacka, Anna; Mozer-Lisewska, Iwona; Koczorowska, Maria

2015-01-01

287

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon alpha and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells.  

PubMed

Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon gamma (IFNgamma), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3+CD56+ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNalpha but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis(Y) antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. PMID:11043851

Flieger, D; Kufer, P; Beier, I; Sauerbruch, T; Schmidt-Wolf, I G

2000-10-01

288

Urokinase expression by tumor suppressor protein p53: a novel role in mRNA turnover.  

PubMed

Lung carcinoma (H1299) cells deficient in p53 (p53(-/-)) express large amounts of urokinase-type plasminogen activator (uPA) protein and uPA mRNA, and exhibit slower degradation of uPA mRNA than that of p53-expressing nonmalignant Beas2B human airway epithelial cells. Expression of p53 protein in H1299 cells, upon transfection with p53 cDNA, suppressed basal as well as uPA-induced expression of uPA protein in both conditioned media and cell lysates, and decreased the level of steady-state uPA mRNA primarily due to increased uPA mRNA turnover. Inhibition of p53 expression by RNA silencing (SiRNA) in Beas2B cells enhanced basal and uPA-mediated uPA protein and mRNA expression with stabilization of uPA mRNA. Purified p53 binds to the uPA mRNA 3' untranslated region (UTR) in a sequence-specific manner and endogenous uPA mRNA associates with p53 protein isolated from Beas2B cytosolic extracts. p53 binds to a 35-nucleotide uPA 3'UTR sequence and insertion of this sequence into beta-globin mRNA accelerates degradation of otherwise stable beta-globin mRNA. These observations confirm a new role for p53 as a uPA mRNA binding protein that down-regulates uPA mRNA stability and decreases cellular uPA expression. PMID:18390474

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Shetty, Rashmi S; Liu, Ming-Cheh; Shetty, Sreerama

2008-09-01

289

New Study Shows Protein and mRNA Levels Are Not Correlated | Physical Sciences in Oncology  

Cancer.gov

Ever since molecular biologists sorted out the general mechanisms that cells use to turn DNA into messenger RNA (mRNA) and then into protein, researchers have largely assumed that the amount of mRNA a cell makes during gene transcription will correlate with the subsequent amount of protein the cell produces from that mRNA. Indeed, aggregate measurements made from large numbers of cells have long supported this assumption.

290

Laparotomy Causes a Transient Induction of Rat Liver Serine Dehydratase mRNA  

Microsoft Academic Search

Rat liver serine dehydratase mRNA shows rhythmicity with a high level at the onset of dark (19:00) and a low level at the onset of light (07:00). We have examined the effect of stress (laparotomy) on the rhythm, Upon laparotomy at 09:00 or 17:00, a marked induction of serine dehydratase mRNA occurred 2 h after operation. The elevated mRNA level

H. Ogawa; S. Kawamata; T. Gomi; Y. Ansai; Y. Karaki

1995-01-01

291

Multiple Cytokine mRNA Expression in Human Mast Cells Stimulated via FcεRI  

Microsoft Academic Search

Cross-linkage of FcεRI on human lung mast cells purified by affinity magnetic selection with monoclonal antibody YB5.B8 against c-kit (purity > 90%) expressed mRNA for multiple cytokines. There was no constitutive expression of interleukin (IL)-4 mRNA. Mast cell stimulation with anti-IgE induced IL-4 mRNA expression which appeared maxmal at 2 h and waned slowly over the next 24 h. IL-5,

Y. Okayama; A. Semper; S. T. Holgate; M. K. Church

1995-01-01

292

Functional Link between the Mammalian Exosome and mRNA Decapping  

Microsoft Academic Search

Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3? to 5? exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the

Zuoren Wang; Megerditch Kiledjian

2001-01-01

293

Metal and Tissue-Dependent Relationship between Metallothionein mRNA and Protein  

Microsoft Academic Search

Metallothionein (MT) expression is transcriptionally regulated but recent evidence suggests that translation of MT mRNA may be regulated under some circumstances (Vasconcelos et al., Biochem. J., 315, 665–671, 1996). A systematic investigation of MT mRNA, protein, and metal levels in liver and kidney of cadmium- or copper-treated rats was made to further understand the relationship between mRNA and protein in

M. Helena Vasconcelos; Shuk-Ching Tam; John E. Hesketh; Martin Reid; John H. Beattie

2002-01-01

294

Detection of bcrabl fusion mRNA in samples of whole, unfractionated blood.  

PubMed

Using a new strategy, capture-RT-PCR, bcrabl fusion mRNA sequences were specifically and sensitively detected in samples of whole blood from leukemia patients with the Philadelphia chromosome. Sample processing required only mixing blood with the chaotropic salt, GuSCN, and mRNA was captured during a short incubation of prepared blood with an affinity membrane. Immobilized mRNA sequences were amplified without elution. No radioisotopes or Southern transfer were needed. PMID:8207964

Kolbe, T; Cuddy, K; Brodsky, I; Marks, D I; Gillespie, D

1994-06-01

295

Structural and functional features of eukaryotic mRNA untranslated regions  

Microsoft Academic Search

The crucial role of the non-coding portion of genomes is now widely acknowledged. In particular, mRNA untranslated regions are involved in many post-transcriptional regulatory pathways that control mRNA localization, stability and translation efficiency. We review in this paper the major structural and compositional features of eukaryotic mRNA untranslated regions and provide some examples of bioinformatic analyses for their functional characterization.

Graziano Pesole; Flavio Mignone; Carmela Gissi; Giorgio Grillo; Flavio Licciulli; Sabino Liuni

2001-01-01

296

Control of the mRNA for Hepatic Tryptophan Oxygenase during Hormonal and Substrate Induction  

Microsoft Academic Search

Glucocorticoid hormones increase the level of hepatic tryptophan oxygenase [EC 1.13.11.11; L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing)] by increasing its rate of synthesis. Studies were performed to determine whether this induction is mediated by controlling the level of the mRNA for tryptophan oxygenase or by changing the translational efficiency of a fixed level of mRNA. Activity of tryptophan oxygenase mRNA was quantitated in

G. Schutz; L. Killewich; G. Chen; P. Feigelson

1975-01-01

297

ORIGINAL PAPER Discovery of potent inhibitors for interleukin-2-inducible  

E-print Network

an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model (HBD), one ring ar- omatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 homology2 DS Discovery Studio v.2.5 HBA Hydrogen-bond acceptor HBD Hydrogen-bond donor HY Hydrophobic R

Lee, Keun Woo

298

ISSN 1360-1725 Mathematical Modelling Of The Interleukin-2  

E-print Network

are key issues in immunology, tu- mour growth and cell biology. We study the kinetics of cell growth T-cells ­ the model of which can be considered to be a general model of cell growth. We also review proliferation, is a central topic in cell biology, immunology and tumour growth. Historically, ODEs have been

Paul, Christopher A.H.

299

ISSN 1360--1725 Mathematical Modelling Of The Interleukin2  

E-print Network

phenomena are key issues in immunology, tu­ mour growth and cell biology. We study the kinetics of cell. We also review the numerical techniques available for solving delay differential equations, immunology and tumour growth. Historically, ODEs have been used to model cell growth -- this is mainly due

Higham, Nicholas J.

300

TOPICAL REVIEW: Mechanisms governing the control of mRNA translation  

NASA Astrophysics Data System (ADS)

The translation of cellular mRNA to protein is a tightly controlled process often deregulated in diseases such as cancer. Furthering our understanding of mRNA structural elements and the intracellular proteins and signaling pathways that affect protein expression is crucial in the development of new therapies. In this review, we discuss the current state-of-the-art of detecting and determining the role of mRNA sequence elements in regulating the initiation of mRNA translation and the therapeutic strategies that exploit this knowledge to treat disease.

Livingstone, Mark; Atas, Evrim; Meller, Amit; Sonenberg, Nahum

2010-06-01

301

Intracellular Calcium Regulates Nonsense-Mediated mRNA Decay  

PubMed Central

The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons (PTCs) and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent an important target for therapeutic intervention. Here we have developed a novel multicolored, bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides including ouabain and digoxin as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the Na+/K+-ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has important implications for exploiting NMD in the treatment of disease. PMID:25064126

Nickless, Andrew; Jackson, Erin; Marasa, Jayne; Nugent, Patrick; Mercer, Robert W.; Piwnica-Worms, David; You, Zhongsheng

2014-01-01

302

Metabolite sensing in eukaryotic mRNA biology.  

PubMed

All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-messenger systems. More recent studies reveal that metabolites also affect post-transcriptional regulatory mechanisms. Here, we review the increasing number of connections between metabolism and post-transcriptional regulation in eukaryotic organisms. First, we present evidence that riboswitches, a common mechanism of metabolite sensing in bacteria, also function in eukaryotes. Next, we review an example of a double stranded RNA modifying enzyme that directly interacts with a metabolite, suggesting a link between RNA editing and metabolic state. Finally, we discuss work that shows some metabolic enzymes bind directly to RNA to affect mRNA stability or translation efficiency. These examples were discovered through gene-specific genetic, biochemical, and structural studies. A directed systems level approach will be necessary to determine whether they are anomalies of evolution or pioneer discoveries in what may be a broadly connected network of metabolism and post-transcriptional regulation. PMID:23653333

Clingman, Carina C; Ryder, Sean P

2013-01-01

303

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress  

Microsoft Academic Search

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-d-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was

Mukaila A Akinbami; Gundala H Philip; Rajagopala Sridaran; Virendra B Mahesh; David R Mann

1999-01-01

304

Following Temperature Stress, Export of Heat Shock mRNA Occurs Efficiently in Cells with Mutations in Genes Normally Important for mRNA Export  

Microsoft Academic Search

Heat shock leads to accumulation of polyadenylated RNA in nuclei of Saccharomyces cerevisiae cells, tran- scriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect SSA4 mRNA (encoding Hsp70) and flow cytometry to measure

Christiane Rollenhagen; Christine A. Hodge; Charles N. Cole

2007-01-01

305

In vitro Splicing of Influenza Viral NS1 mRNA and NS1-? -globin Chimeras: Possible Mechanisms for the Control of Viral mRNA Splicing  

NASA Astrophysics Data System (ADS)

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human ? -globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and ? -globin sequences, we show that the NS1 5' splice site was effectively utilized by the ? -globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the ? -globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from ? -globin With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

Plotch, Stephen J.; Krug, Robert M.

1986-08-01

306

Interactions of Elongation Factor 1 with FActin and Actin mRNA: Implications for Anchoring mRNA in Cell Protrusions  

Microsoft Academic Search

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin\\/ microtubule-binding protein, elongation factor 1 (EF1) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1 colocalizes with -actin mRNA and F-actin

Gang Liu; Wayne M. Grant; Daniel Persky; Vaughan M. Latham; Robert H. Singer; John Condeelis

307

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription  

PubMed Central

Objective Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific mRNA in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. Methods/Results Murine bone marrow cells co-cultured with rat lung, but separated from them using a cell-impermeable membrane (0.4 micron pore size), were analyzed using species-specific primers (for rat or mouse). These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung co-cultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after co-culture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and also mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer term stable change in genetic phenotype which has been observed. We have also observed microRNA in lung-derived microvesicles and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in co-cultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart and liver mRNA in co-cultured marrow cells suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. Conclusion These studies suggest that cellular systems are more phenotypically labile then previously considered. PMID:20079801

Aliotta, Jason M.; Pereira, Mandy; Johnson, Kevin W.; de Paz, Nicole; Dooner, Mark S.; Puente, Napoleon; Ayala, Carol; Brilliant, Kate; Berz, David; Lee, David; Ramratnam, Bharat; McMillan, Paul N.; Hixson, Douglas C.; Josic, Djuro; Quesenberry, Peter J.

2010-01-01

308

Distinguishing direct from indirect roles for bicoid mRNA localization factors  

PubMed Central

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. Here we use live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. We show that Swallow and bicoid mRNA are transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, and we present evidence that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. Our results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead we propose a model for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport. PMID:20023172

Weil, Timothy T.; Xanthakis, Despina; Parton, Richard; Dobbie, Ian; Rabouille, Catherine; Gavis, Elizabeth R.; Davis, Ilan

2010-01-01

309

Degradation of intestinal mRNA: A matter of treatment  

PubMed Central

AIM: To characterize the influence of location, species and treatment upon RNA degradation in tissue samples from the gastrointestinal tract. METHODS: The intestinal samples were stored in different medium for different times under varying conditions: different species (human and rat), varying temperature (storage on crushed ice or room temperature), time point of dissection of the submucous-mucous layer from the smooth muscle (before or after storage), different rinsing methods (rinsing with Medium, PBS, RNALater or without rinsing at all) and different regions of the gut (proximal and distal small intestine, caecum, colon and rectum). The total RNA from different parts of the gut (rat: proximal and distal small intestine, caecum, colon and rectum, human: colon and rectum) and individual gut layers (muscle and submucosal/mucosal) was extracted. The quality of the RNA was assessed by micro capillary electrophoresis. The RNA quality was expressed by the RNA integrity number which is calculated from the relative height and area of the 18 S and 28 S RNA peaks. From rat distal small intestine qPCR was performed for neuronal and glial markers. RESULTS: RNA obtained from smooth muscle tissue is much longer stable than those from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, on ice it is stable at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. RNA obtained from the submucosal/mucosal layer always showed a much worse amplification rate than RNA from muscle tissue. In general RNA harvested from rat tissue, either smooth muscle layer or submucosal/mucosal layer is much longer stable than RNA from human gut tissue, and RNA obtained from smooth muscle tissue shows an increased stability compared to RNA from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, while the stability on ice lasts at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. The RNA from muscle and submucosal/mucosal tissue of the proximal small intestine degrades much faster than the RNA of distal small intestine, caecum or colon with rectum. RNA obtained from the submucosal/mucosal layer always showed a much more reduced amplification rate than RNA from muscle tissue [?-Tubulin III for muscle quantification cycle (Cp): 22.07 ± 0.25, for ?-Tubulin III submucosal/mucosal Cp: 27.42 ± 0.19]. CONCLUSION: Degradation of intestinal mRNA depends on preparation and storage conditions of the tissue. Cooling, rinsing and separating of intestinal tissue reduce the degradation of mRNA.

Heumüller-Klug, Sabine; Sticht, Carsten; Kaiser, Karin; Wink, Elvira; Hagl, Cornelia; Wessel, Lucas; Schäfer, Karl-Herbert

2015-01-01

310

Differentiation equations III: mRNA transcription-protein translation model  

NSDL National Science Digital Library

The first video segment presents a canonical mathematical example from quantitative biology, in which mRNA is transcribed from a gene sequence, and protein is translated from mRNA. The second segment uses eigenvector-eigenvalue analysis to sketch the trajectories of the system in a phase portrait. Finally, the third segment generalizes the linear stability analysis used to study this example.

2013-06-21

311

GALLUS GALLUS PREPROGLUCAGON (GCG), CLASS B MRNA, TRANSCRIPT VARIANT 1, COMPLETE EDS  

Technology Transfer Automated Retrieval System (TEKTRAN)

The proglucagon gene in mammals produces a single identical mRNA transcript in all tissues where it is expressed. This transcript encodes glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2). Previously, two distinct proglucagon mRNA transcripts have been identified in chickens, one in...

312

Regulation of the mRNA half-life in breast cancer  

PubMed Central

The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3’UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

Griseri, Paola; Pagès, Gilles

2014-01-01

313

Presence of mRNA for Glutamic Acid Decarboxylase in both Excitatory and Inhibitory Neurons  

Microsoft Academic Search

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma -aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD

Yanxiang Cao; Karen S. Wilcox; Claudia E. Martin; Tara L. Rachinsky; James Eberwine; Marc A. Dichter

1996-01-01

314

Profiling condition-specific, genome-wide regulation of mRNA stability in yeast  

E-print Network

. Sean Houshmandi , Wendy M. Olivas , and Harmen J. Bussemaker*§ *Department of Biological SciencesProfiling condition-specific, genome-wide regulation of mRNA stability in yeast Barrett C. Foat*, S May 8, 2005) The steady-state abundance of an mRNA is determined by the balance between transcription

Olivas, Wendy M.

315

Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology  

PubMed Central

Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3? UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3? extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3–aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis. PMID:24889622

Michaud, Morgane; Ubrig, Elodie; Filleur, Sophie; Erhardt, Mathieu; Ephritikhine, Geneviève; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-01-01

316

GALLUS GALLUS PREPROGLUCAGON (GCG), CLASS A MRNA, TRANSCRIPT VARIANT 1, COMPLETE CDS  

Technology Transfer Automated Retrieval System (TEKTRAN)

In mammals, the proglucagon gene produces a single identical mRNA transcript in pancreas and intestine. This transcript encodes glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2). Previously, two distinct proglucagon mRNA transcripts have been identified in chickens, one in pancreas ...

317

Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability  

Microsoft Academic Search

The metabolism of eukaryotic mRNA occurs in both the nucleus and cytoplasm and involves a coordinated balance between transcriptional and posttranscrip- tional events. Posttranscriptional regulation can occur at many stages in the processing of a transcript, in- cluding its demise. A major contribution to the post- transcriptional regulation of mammalian gene expression is at the level of mRNA stability. The

BETSY T. KREN; CLIFFORD J. STEER

318

Deciphering molecular mechanisms of mRNA metabolism in the deep-branching eukaryote Entamoeba histolytica.  

PubMed

Although extraordinary rapid advance has been made in the knowledge of mechanisms regulating messenger RNA (mRNA) metabolism in mammals and yeast, little information is known in deep-branching eukaryotes. The complete genome sequence of Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, provided a lot of information for the identification and comparison of regulatory sequences and proteins potentially involved in mRNA synthesis, processing, and degradation. Here, we review the current knowledge of mRNA metabolism in this human pathogen. Several DNA motifs in promoter and nuclear factors involved in transcription, as well as conserved polyadenylation sequences in mRNA 3'-untranslated region and possible cleavage and polyadenylation factors, are described. In addition, we present recent data about proteins involved in mRNA decay with a special focus on the recently reported P-bodies in amoeba. Models for mechanisms of decapping and deadenylation-dependent pathways are discussed. We also review RNA-based gene silencing mechanisms and describe the DEAD/DExH box RNA helicases that are molecular players in all mRNA metabolism reactions. The functional characterization of selected proteins allows us to define a general framework to describe how mRNA synthesis, processing, and decay may occur in E. histolytica. Taken altogether, studies of mRNA metabolism in this single-celled eukaryotic model suggest the conservation of specific gene expression regulatory events through evolution. PMID:24249245

López-Camarillo, César; López-Rosas, Itzel; Ospina-Villa, Juan David; Marchat, Laurence A

2014-01-01

319

Nova Mediates Experience Dependent Processing of Orb2A mRNA  

E-print Network

the Drosophila homologue of Nova-1/2, a well characterized mammalian nervous system specific alternative splicing factor. Our results implicate mRNA processing as a regulatory step in memory formation via the Nova dependent maturation of Orb2A mRNA....

Gill, Jason Singh

2013-08-31

320

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.  

PubMed

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

321

Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA  

PubMed Central

Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA. PMID:21468235

Forget, Amélie

2011-01-01

322

Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human  

E-print Network

Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human micro assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for ,8,000 genes in these proliferative

Herschlag, Dan

323

Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR  

Technology Transfer Automated Retrieval System (TEKTRAN)

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

324

RT-isoPCR: nested, high multiplex mRNA amplification.  

PubMed

RT-isoPCR provides multiplex amplification of mRNA targets using a first-stage multiplex RT-PCR reaction with subsequent isothermal amplification for individual target loci detection. We demonstrate detection of 24 mRNA targets with high specificity and sensitivity without compromising sample variation or introducing biases between targets. PMID:23964356

Søe, Martin Jensen; Warthoe, Peter

2013-10-21

325

PROLONGED FASTING AND CORTISOL REDUCE MYOSTATIN MRNA LEVELS IN TILAPIA LARVAE, SHORT-TERM FASTING ELEVATES  

Technology Transfer Automated Retrieval System (TEKTRAN)

Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indices in adults, skeletal muscle myostatin mRNA...

326

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient  

PubMed Central

Summary The Bicoid (Bcd) protein gradient is generally believed to be established in pre-blastoderm Drosophila embryos by the diffusion of Bcd protein after translation of maternal mRNA, which serves as a strictly localized source of Bcd at the anterior pole. However, we previously published evidence that the Bcd gradient is preceded by a bcd mRNA gradient. Here, we have revisited and extended this observation by showing that the bcd mRNA and Bcd protein gradient profiles are virtually identical at all times. This confirms our previous conclusion that the Bcd gradient is produced by a bcd mRNA gradient rather than by diffusion. Based on our observation that bcd mRNA colocalizes with Staufen (Stau), we propose that the bcd mRNA gradient forms by a novel mechanism involving quasi-random active transport of a Stau-bcd mRNA complex through a nonpolar microtubular network, which confines the bcd mRNA to the cortex of the embryo. PMID:19168676

Spirov, Alexander; Fahmy, Khalid; Schneider, Martina; Frei, Erich; Noll, Markus; Baumgartner, Stefan

2009-01-01

327

The Regulation of mRNA Stability in Mammalian Cells: 2.0  

PubMed Central

Messenger RNA decay is an essential step in gene expression to set mRNA abundance in the cytoplasm. The binding of proteins and/or noncoding RNAs to specific recognition sequences or secondary structures within mRNAs dictates mRNA decay rates by recruiting specific enzyme complexes that perform the destruction processes. Often, the cell coordinates the degradation or stabilization of functional subsets of mRNAs encoding proteins collectively required for a biological process. As well, extrinsic or intrinsic stimuli activate signal transduction pathways that modify the mRNA decay machinery with consequent effects on decay rates and mRNA abundance. This review is an update to our 2001 Gene review on mRNA stability in mammalian cells, and we survey the enormous progress made over the past decade. PMID:22452843

Wu, Xiangyue; Brewer, Gary

2012-01-01

328

Substance P mRNA expression during zebrafish development: influence of mu opioid receptor and cocaine.  

PubMed

Zebrafish has emerged as an important vertebrate animal model for the study of human diseases and for developmental studies in mammals. Since there are few studies of the tachykinin 1 gene (TAC1), precursor of substance P (SP), in relation to embryonic development, we aimed to study the expression of SP transcript (mRNA) and determine the influence of cocaine and opioid receptors on the expression of this neuropeptide. In order to analyse the spatial and temporal SP mRNA expression in zebrafish, we cloned - based on human TAC1 sequence - the sequence that originates SP. Phylogenetic analyses of the precursor of SP, revealed an alignment in the fish cluster, with a clear distinction from other species (amphibians, birds and mammals). Real time PCR (qPCR) results showed that SP mRNA was expressed in several stages of embryonic development, where it increased progressively from gastrula-8hpf (hour post-fertilisation) to the end of the embryogenesis-72hpf. SP mRNA was expressed mainly in the spinal cord in embryos at 20-30hpf, whereas at 36, 42 and 48hpf embryos SP mRNA was expressed mainly in the CNS telencephalon, diencephalon, hypothalamus, rhombomeres, epiphysis and in peripheral areas (heart and somites). Exposure of embryos to 1.5?M cocaine altered the SP mRNA expression at 24 (increasing) and 48hpf (decreasing). We also report that knockdown of ?-opioid receptor induced an increase of SP mRNA expression while the knockdown of the two delta opioid receptors did not produce changes in SP mRNA expression. In conclusion, SP mRNA in zebrafish is expressed during embryonic development in the CNS and peripherally, suggesting that SP would play a critical role during embryogenesis. Furthermore, cocaine exposure and the knockdown of ?-opioid receptor affect the SP mRNA expression. These observations can be important in the pain and addiction field where SP is involved. PMID:23528978

López-Bellido, Roger; Barreto-Valer, Katherine; Rodríguez, Raquel Emilia

2013-07-01

329

Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles.  

PubMed

Sensory nerve disorders are difficult to cure completely considering poor nerve regeneration capacity and difficulties in accurately targeting neural tissues. Administering mRNA is a promising approach for treating neurological disorders because mRNA can provide proteins and peptides in their native forms for mature non-dividing neural cells, without the need of entering their nuclei. However, direct mRNA administration into neural tissues in vivo has been challenging due to too unstable manner of mRNA and its strong immunogenicity. Thus, using a suitable carrier is essential for effective mRNA administration. For this purpose, we established a novel carrier based on the self-assembly of polyethylene glycol (PEG)-polyamino acid block copolymer, i.e. polyplex nanomicelles. To investigate the feasibility and efficacy of mRNA administration for the treatment of sensory nerve disorders, we used a mouse model of experimentally induced olfactory dysfunction. Intranasal administration of mRNA-loaded nanomicelles provided an efficient and sustained protein expression for nearly two days in nasal tissues, particularly in the lamina propria which contains olfactory nerve fibers, with effectively regulating the immunogenicity of mRNA. Consequently, once-daily intranasal administration of brain-derived neurotrophic factor (BDNF)-expressing mRNA using polyplex nanomicelles remarkably enhanced the neurological recovery of olfactory function along with repairing the olfactory epithelium to a nearly normal architecture. To the best of our knowledge, this is the first study to show the therapeutic potential of introducing exogenous mRNA for the treatment of neurological disorders. These results indicate the feasibility and safety of using mRNA, and provide a novel strategy of mRNA-based therapy. PMID:25599855

Baba, Miyuki; Itaka, Keiji; Kondo, Kenji; Yamasoba, Tatsuya; Kataoka, Kazunori

2015-03-10

330

Structural organization of mRNA complexes with major core mRNP protein YB-1.  

PubMed

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA-YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600-700 nt mRNA segment on its surface. PMID:15494450

Skabkin, Maxim A; Kiselyova, Olga I; Chernov, Konstantin G; Sorokin, Alexey V; Dubrovin, Evgeniy V; Yaminsky, Igor V; Vasiliev, Victor D; Ovchinnikov, Lev P

2004-01-01

331

Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5? to 3? and 3? to 5?  

PubMed Central

Histone mRNAs are rapidly degraded at the end of S phase or when DNA replication is inhibited. Histone mRNAs end in a conserved stem–loop rather than a poly(A) tail. Degradation of histone mRNAs requires the stem–loop sequence, which binds the stem–loop-binding protein (SLBP), active translation of the histone mRNA, and the location of the stem–loop close to the termination codon. We report that the initial step in histone mRNA degradation is the addition of uridines to the 3? end of the histone mRNA, both after inhibition of DNA replication and at the end of S phase. Lsm1 is required for histone mRNA degradation and is present in a complex containing SLBP on the 3? end of histone mRNA after inhibition of DNA replication. We cloned degradation intermediates that had been partially degraded from both the 5? and the 3? ends. RNAi experiments demonstrate that both the exosome and 5?-to-3? decay pathway components are required for degradation, and individual histone mRNAs are then degraded simultaneously 5? to 3? and 3? to 5?. PMID:18172165

Mullen, Thomas E.; Marzluff, William F.

2008-01-01

332

Arc mRNA docks precisely at the base of individual dendritic spines indicating the existence of a specialized microdomain for synapse-specific mRNA translation.  

PubMed

Arc (aka Arg 3.1) is induced by neural activity and learning experience. Arc mRNA is rapidly exported into dendrites where it localizes near activated synapses. By imaging green fluorescent protein (GFP)-tagged mRNA in living neurons in culture, we show that fusion transcripts containing the Arc 30'UTR (untranslated region) localize with remarkable precision in a microdomain at the base of dendritic spines. Transcripts with the Arc 30'UTR that encode a reporter protein rather than Arc show precise localization. Localization persists in the presence of translation inhibitors, indicating that localization does not require ongoing translation. Similarly, polyribosome complexes remained stably positioned at spine bases in brain tissue treated with the translation inhibitor (puromycin) that releases ribosomes from mRNA. Single particle tracking revealed that Arc mRNA particles positioned at spine bases exhibited highly constrained submicron movements. These observations imply the existence of a microdomain at the spine base where Arc mRNA docks in association with a previously unknown mRNA-binding structural element. PMID:22350812

Dynes, Joseph L; Steward, Oswald

2012-10-01

333

Experiment K-6-11. Actin mRNA and cytochrome c mRNA concentrations in the tricepts brachia muscle of rats  

NASA Technical Reports Server (NTRS)

It is well known that some skeletal muscles atrophy as a result of weightlessness (Steffen and Musacchia 1986) and as a result of hindlimb suspension (Tischler et al., 1985, Thomason et al., 1987). Because the content of protein is determined by the rates of protein synthesis and degradation, a decrease in protein synthesis rate, or an increase in the protein degradation, or changes in both could produce the atrophy. Indeed, an increased protein degradation (Tischler et al., 1985) and a decreased protein synthesis (Thomason et al., 1988) have been observed in skeletal muscles of suspended hindlimbs of rats. Any decrease in protein synthesis rate could be caused by decreases in mRNA concentrations. Such decreases in the concentration and content of alpha-actin mRNA and cytochrome c mRNA have been noted in skeletal muscles of hindlimb suspended rats (Babij and Booth, 1988). From these findings researchers hypothesized that alpha-actin mRNA and cytochrome c mRNA would decrease in the triceps brachia muscle of Cosmos 1887 rats.

Booth, F. W.; Morrison, P. R.; Thomason, D. B.; Oganov, V. S.

1990-01-01

334

Impact of STAT/SOCS mRNA Expression Levels after Major Injury  

PubMed Central

Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0?h–72?h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6?h after trauma. SOCS 1 levels significantly decreased 6?h–72?h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6?h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6?h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

2014-01-01

335

Transcription Control Pathways Decode Patterned Synaptic Inputs into Diverse mRNA Expression Profiles  

PubMed Central

Synaptic plasticity requires transcription and translation to establish long-term changes that form the basis for long term memory. Diverse stimuli, such as synaptic activity and growth factors, trigger synthesis of mRNA to regulate changes at the synapse. The palette of possible mRNAs is vast, and a key question is how the cell selects which mRNAs to synthesize. To address this molecular decision-making, we have developed a biochemically detailed model of synaptic-activity triggered mRNA synthesis. We find that there are distinct time-courses and amplitudes of different branches of the mRNA regulatory signaling pathways, which carry out pattern-selective combinatorial decoding of stimulus patterns into distinct mRNA subtypes. Distinct, simultaneously arriving input patterns that impinge on the transcriptional control network interact nonlinearly to generate novel mRNA combinations. Our model combines major regulatory pathways and their interactions connecting synaptic input to mRNA synthesis. We parameterized and validated the model by incorporating data from multiple published experiments. The model replicates outcomes of knockout experiments. We suggest that the pattern-selectivity mechanisms analyzed in this model may act in many cell types to confer the capability to decode temporal patterns into combinatorial mRNA expression. PMID:24787753

Jain, Pragati; Bhalla, Upinder S.

2014-01-01

336

Definition of global and transcript-specific mRNA export pathways in metazoans  

PubMed Central

Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms’ export pathways. Here we report the results of a genome-wide RNAi screen in which we identify 72 factors required for polyadenylated [poly-(A+)] mRNA export from the nucleus in Drosophila cells. Using structural and functional conservation analysis of yeast and Drosophila mRNA export factors, we expose the evolutionary divergence of eukaryotic mRNA export pathways. Additionally, we demonstrate the differential export requirements of two endogenous heat-inducible transcripts—intronless heat-shock protein 70 (HSP70) and intron-containing HSP83—and identify novel export factors that participate in HSP83 mRNA splicing. We characterize several novel factors and demonstrate their participation in interactions with known components of the Drosophila export machinery. One of these factors, Drosophila melanogaster PCI domain-containing protein 2 (dmPCID2), associates with polysomes and may bridge the transition between exported messenger ribonucleoprotein particles (mRNPs) and polysomes. Our results define the global network of factors involved in Drosophila mRNA export, reveal specificity in the export requirements of different transcripts, and expose new avenues for future work in mRNA export. PMID:18086857

Farny, Natalie G.; Hurt, Jessica A.; Silver, Pamela A.

2008-01-01

337

Photoregulation of ?-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings 1  

PubMed Central

The effect of light on the abundance of ?-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat ?-tubulin cDNA clone was used to measure ?-tubulin mRNA levels. White light induced a 45% decrease in oat ?-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in ?-tubulin mRNA levels in oats and barley, respectively. Recovery of ?-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat ?-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in ?-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the ?-tubulin genes. Images Figure 1 Figure 2 PMID:16667578

Colbert, James T.; Costigan, Stephen A.; Zhao, Zhifan

1990-01-01

338

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization.  

PubMed

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport. PMID:18775316

Zimyanin, Vitaly L; Belaya, Katsiaryna; Pecreaux, Jacques; Gilchrist, Michael J; Clark, Alejandra; Davis, Ilan; St Johnston, Daniel

2008-09-01

339

Context effects on N6-adenosine methylation sites in prolactin mRNA.  

PubMed Central

The methylation of internal adenosine residues in mRNA only occurs within GAC or AAC sequences. Although both of these sequence motifs are utilized, a general preference has been noted for the extended sequence RGACU. Not all RGACU sequences in an mRNA are methylated and the mechanisms that govern the selection of methylation sites in mRNA remain unclear. To address this problem we have examined the methylation of transcripts containing sequences of a natural mRNA, namely, bovine prolactin mRNA. In this mRNA, a specific AGACU sequence in the 3' untranslated region is the predominant site of methylation both in vivo and in vitro. The degree to which N6-adenosine methyltransferase recognizes the sequence context of the consensus methylation site was explored by mutational analysis of the nucleotides adjacent to the core sequence as well as the extended regions in which the core element was found. Our results indicate that efficient methylation depends on the extended five nucleotide consensus sequence but is strongly influenced by the context in which the consensus sequence occurs within the overall mRNA molecule. Furthermore, consensus methylation sites present in an RNA duplex are not recognized by the methyltransferase. Images PMID:8127679

Narayan, P; Ludwiczak, R L; Goodwin, E C; Rottman, F M

1994-01-01

340

A nucleic acid biosensor for gene expression analysis in nanograms of mRNA.  

PubMed

An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels. PMID:15253638

Xie, Hong; Yu, Yuan Hong; Xie, Fang; Lao, Yuan Zhi; Gao, Zhiqiang

2004-07-15

341

The protein Mago provides a link between splicing and mRNA localization  

PubMed Central

The proteins Mago and Y14 are evolutionarily conserved binding partners. Y14 is a component of the exon–exon junction complex (EJC), deposited by the spliceosome upstream of messenger RNA (mRNA) exon–exon junctions. The EJC is implicated in post-splicing events such as mRNA nuclear export and nonsense-mediated mRNA decay. Drosophila Mago is essential for the localization of oskar mRNA to the posterior pole of the oocyte, but the functional role of Mago in other species is unknown. We show that Mago is a bona fide component of the EJC. Like Y14, Mago escorts spliced mRNAs to the cytoplasm, providing a direct functional link between splicing and the downstream process of mRNA localization. Mago/Y14 heterodimers are essential in cultured Drosophila cells. Taken together, these results suggest that, in addition to its specialized function in mRNA localization, Mago plays an essential role in other steps of mRNA metabolism. PMID:11743026

Le Hir, Hervé; Gatfield, David; Braun, Isabelle C.; Forler, Daniel; Izaurralde, Elisa

2001-01-01

342

The protein Mago provides a link between splicing and mRNA localization.  

PubMed

The proteins Mago and Y14 are evolutionarily conserved binding partners. Y14 is a component of the exon-exon junction complex (EJC), deposited by the spliceosome upstream of messenger RNA (mRNA) exon-exon junctions. The EJC is implicated in post-splicing events such as mRNA nuclear export and nonsense-mediated mRNA decay. Drosophila Mago is essential for the localization of oskar mRNA to the posterior pole of the oocyte, but the functional role of Mago in other species is unknown. We show that Mago is a bona fide component of the EJC. Like Y14, Mago escorts spliced mRNAs to the cytoplasm, providing a direct functional link between splicing and the downstream process of mRNA localization. Mago/Y14 heterodimers are essential in cultured Drosophila cells. Taken together, these results suggest that, in addition to its specialized function in mRNA localization, Mago plays an essential role in other steps of mRNA metabolism. PMID:11743026

Le Hir, H; Gatfield, D; Braun, I C; Forler, D; Izaurralde, E

2001-12-01

343

Single-molecule modeling of mRNA degradation by miRNA: Lessons from data  

E-print Network

Recent experimental results on the effect of miRNA on the decay of its target mRNA have been analyzed against a previously hypothesized single molecule degradation pathway. According to that hypothesis, the silencing complex (miRISC) first interacts with its target mRNA and then recruits the protein complexes associated with NOT1 and PAN3 to trigger deadenylation (and subsequent degradation) of the target mRNA. Our analysis of the experimental decay patterns allowed us to refine the structure of the degradation pathways at the single molecule level. Surprisingly, we found that if the previously hypothesized network was correct, only about 7% of the target mRNA would be regulated by the miRNA mechanism, which is inconsistent with the available knowledge. Based on systematic data analysis, we propose the alternative hypothesis that NOT1 interacts with miRISC before binding to the target mRNA. Moreover, we show that when miRISC binds alone to the target mRNA, the mRNA is degraded more slowly, probably through a ...

Sin, Celine; Valleriani, Angelo

2014-01-01

344

c-myc mRNA expression in non-Hodgkin's lymphomas.  

PubMed

Steady state c-myc mRNA levels determined by Northern blot analysis were examined in non-Hodgkin's lymphomas (NHL) of both high (n = 29) and low malignancy (n = 18), and in non-specific chronic lymphadenitis (n = 6). High grade NHL, classified according to the updated Kiel classification, revealed significantly larger amounts of c-myc mRNA compared with low grade NHL and lymphadenitis. mRNA levels in non-specific lymphadenitis were lower than in low grade NHL, but the differences were not statistically significant. No correlation between c-myc mRNA levels and the immunologic phenotype was discernible. Growth fractions of the NHL were determined by immunostaining with the monoclonal antibody Ki-67. Significant correlations between the percentages of Ki-67-positive cells, as well as the amounts of c-myc mRNA, and classification into high or low grade NHL were found. However, the percentage of Ki-67 positive cells and c-myc mRNA levels in individual cases and in the various histologic entities of NHL did not correlate. Our results indicate the overexpression of the c-myc gene in NHL, and a highly significant correlation of steady state c-myc mRNA levels with the prognosis-related histomorphologic Kiel classification of NHL into different subgroups of low and high grade malignancy. PMID:1352077

Fellbaum, C; Radaszkiewicz, T; Ruhri, C; Pütz, B; Lehmacher, W; Höfler, H

1992-01-01

345

Single-molecule modeling of mRNA degradation by miRNA: Lessons from data  

E-print Network

Recent experimental results on the effect of miRNA on the decay of its target mRNA have been analyzed against a previously hypothesized single molecule degradation pathway. According to that hypothesis, the silencing complex (miRISC) first interacts with its target mRNA and then recruits the protein complexes associated with NOT1 and PAN3 to trigger deadenylation (and subsequent degradation) of the target mRNA. Our analysis of the experimental decay patterns allowed us to refine the structure of the degradation pathways at the single molecule level. Surprisingly, we found that if the previously hypothesized network was correct, only about 7% of the target mRNA would be regulated by the miRNA mechanism, which is inconsistent with the available knowledge. Based on systematic data analysis, we propose the alternative hypothesis that NOT1 interacts with miRISC before binding to the target mRNA. Moreover, we show that when miRISC binds alone to the target mRNA, the mRNA is degraded more slowly, probably through a deadenylation-independent pathway. The new biochemical pathway we propose both fits the data and paves the way for new experimental work to identify new interactions.

Celine Sin; Davide Chiarugi; Angelo Valleriani

2014-10-20

346

Isolation and Amplification of mRNA within a Simple Microfluidic Lab on a Chip  

PubMed Central

The major modules for realizing molecular biological assays in a micro total analysis system (?TAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic messenger RNA (mRNA) within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid non-specific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 1015 fold and still result in successful on-chip re-amplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to bench-top devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes. PMID:24328414

Reinholt, Sarah J.; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J.

2014-01-01

347

Expression and stability of c-sis mRNA in human glioblastoma cells  

SciTech Connect

The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, the authors have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.

Press, R.D.; Samols, D.; Goldthwait, D.A.

1988-07-26

348

Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity  

PubMed Central

Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

2014-01-01

349

Increased expression of growth hormone receptor mRNA and insulin-like growth factor-I mRNA in volume-overloaded hearts.  

PubMed

Recent results suggest that insulin-like growth factor-I (IGF-I) may be involved in the transition of a hemodynamic load into cardiac hypertrophy and that the expression of IGF-I seems to be coupled to increased wall stress. The present study investigated the role of growth hormone (GH) and IGF-I in myocardial hypertrophy induced by volume overload. An aortocaval fistula (ACF) was created in male Wistar rats, and experiments were performed 2, 4, and 7 days after the onset of volume overload. Right and left ventricular (RV and LV, respectively) myocardial expression of GH receptor mRNA and IGF-I mRNA were quantitated by a solution hybridization RNase protection assay. RV GH receptor mRNA content was elevated on the fourth and seventh days after the induction of the shunt, with peak levels (0.63 +/- 0.16 versus 0.14 +/- 0.03 amol/microgram DNA for the sham-operated animals; P < .01) after 4 days. Similarly, IGF-I mRNA was significantly increased in the RV of shunted animals (1.26 +/- 0.13 versus 0.56 +/- 0.05 amol/micrograms DNA; P < .01) 7 days after surgery. In the left ventricle, where systolic pressure was reduced in ACF rats, no differences could be detected in GH receptor and IGF-I mRNA content between ACF and sham-operated rats on any of the experimental days. There was no difference in the ratio of RV to LV weight during the experimental period. We have shown that the thin-walled right ventricle responds to volume overload with an increase of GH receptor mRNA content followed by elevated expression of IGF-I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8206622

Isgaard, J; Wåhlander, H; Adams, M A; Friberg, P

1994-06-01

350

Distinct RNP Complexes of Shuttling hnRNP Proteins with Pre-mRNA and mRNA: Candidate Intermediates in Formation and Export of mRNA  

Microsoft Academic Search

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA

STAVROULA MILI; HONG JUN SHU; YINGMING ZHAO; SERAFIN PINOL-ROMA

2001-01-01

351

Nonsense-mediated mRNA decapping occurs on polyribosomes in Saccharomyces cerevisiae  

PubMed Central

Nonsense-mediated decay (NMD) degrades mRNA containing premature translation termination codons. In yeast, NMD substrates are decapped and digested exonucleolytically from the 5? end. Despite the requirement for translation in recognition, degradation of nonsense-containing mRNA is considered to occur in ribosome-free cytoplasmic P bodies. We show decapped nonsense-containing mRNA associate with polyribosomes, indicating that recognition and degradation are tightly coupled and that polyribosomes are major sites for degradation of aberrant mRNAs. PMID:20118937

Hu, Wenqian; Petzold, Christine; Coller, Jeff; Baker, Kristian E

2010-01-01

352

Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR  

PubMed Central

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

Ko, Gwangpyo; Cromeans, Theresa L.; Sobsey, Mark D.

2003-01-01

353

Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR.  

PubMed

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples. PMID:14660388

Ko, Gwangpyo; Cromeans, Theresa L; Sobsey, Mark D

2003-12-01

354

Luzp4 defines a new mRNA export pathway in cancer cells  

PubMed Central

Cancer testis antigens (CTAs) represented a poorly characterized group of proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells. Here we show that one CTA, Luzp4, is an mRNA export adaptor. It associates with the TREX mRNA export complex subunit Uap56 and harbours a Uap56 binding motif, conserved in other mRNA export adaptors. Luzp4 binds the principal mRNA export receptor Nxf1, enhances its RNA binding activity and complements Alyref knockdown in vivo. Whilst Luzp4 is up-regulated in a range of tumours, it appears preferentially expressed in melanoma cells where it is required for growth. PMID:25662211

Viphakone, Nicolas; Cumberbatch, Marcus G.; Livingstone, Michaela J.; Heath, Paul R.; Dickman, Mark J.; Catto, James W.; Wilson, Stuart A.

2015-01-01

355

Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action  

PubMed Central

Changes in mRNA stability and translation are critical control points in the regulation of gene expression, particularly genes encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenosine and uridine (AU)-rich elements (ARE), often located in the 3? untranslated regions (3?UTR) of mRNAs, are known to target transcripts for rapid decay. They are also involved in the regulation of mRNA stability and translation in response to extracellular cues. This review focuses on one of the best characterized ARE binding proteins, tristetraprolin (TTP), the founding member of a small family of CCCH tandem zinc finger proteins. In this survey, we have reviewed the current status of TTP interactions with mRNA and proteins, and discussed current thinking about TTP's mechanism of action to promote mRNA decay. We also review the proposed regulation of TTP's functions by phosphorylation. Finally, we have discussed emerging evidence for TTP operating as a translational regulator. PMID:23428348

Brooks, Seth A.; Blackshear, Perry J.

2013-01-01

356

Positive and negative feedback loops in the p53 and mRNA 3? processing pathways  

PubMed Central

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. Recent studies have shown regulation of p53 at the mRNA level mediated via both the 5? and the 3? untranslated regions and affecting the stability and translation efficiency of the p53 mRNA. Here, we provide evidence of a feedback loop between p53 and the poly(A)-specific ribonuclease (PARN), in which PARN deadenylase keeps p53 levels low in nonstress conditions by destabilizing p53 mRNA, and the UV-induced increase in p53 activates PARN deadenylase, regulating gene expression during DNA damage response in a transactivation-independent manner. This model is innovative because it provides insights into p53 function and the mechanisms behind the regulation of mRNA 3? end processing in different cellular conditions. PMID:23401530

Devany, Emral; Zhang, Xiaokan; Park, Ji Yeon; Tian, Bin; Kleiman, Frida Esther

2013-01-01

357

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition  

PubMed Central

Background Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. Results The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. Conclusion Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking. PMID:24070093

2013-01-01

358

Premature 39End Formation of CBP1 mRNA Results in the Downregulation of Cytochrome b mRNA during the Induction of Respiration in Saccharomyces cerevisiae  

Microsoft Academic Search

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the

KIMBERLY A. SPARKS; STEPHEN A. MAYER; CAROL L. DIECKMANN

1997-01-01

359

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.  

PubMed

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels. PMID:10622402

Akinbami, M A; Philip, G H; Sridaran, R; Mahesh, V B; Mann, D R

1999-01-01

360

Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases  

PubMed Central

In eukaryotes, shortening of the 3?-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes—Caf1–Ccr4 and Pan2–Pan3—play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1–Ccr4 and Pan2–Pan3. Interestingly, translation termination complexes eRF1–eRF3, Pan2–Pan3, and Caf1–Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1–eRF3 for the deadenylase occurs on PABPC1. Consequently, PABPC1 binding leads to the activation of Pan2–Pan3 and Caf1–Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2–Pan3 and Caf1–Ccr4 in cooperation with eRF3 and PABPC1. PMID:18056425

Funakoshi, Yuji; Doi, Yusuke; Hosoda, Nao; Uchida, Naoyuki; Osawa, Masanori; Shimada, Ichio; Tsujimoto, Masafumi; Suzuki, Tsutomu; Katada, Toshiaki; Hoshino, Shin-ichi

2007-01-01

361

The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.  

PubMed

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. When assembled in vitro, this so-called 'exon-exon junction complex' (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm. PMID:11532962

Le Hir, H; Gatfield, D; Izaurralde, E; Moore, M J

2001-09-01

362

ADAM17 mRNA expression and pathological features of hepatocellular carcinoma  

Microsoft Academic Search

AIM: To study the expression of a disintegrin and metalloproteinase 17 (ADAM17) mRNA in hepatocellular carcinoma (HCC) and to evaluate the relationship between ADAM17 mRNA expression and clinicopathological features of HCC. METHODS: Hepatocellular carcinomas (HCC) from 31 cases were divided into small HCC (SHCC), nodular HCC (NHCC) and solitary large HCC (SLHCC) according to tumor diameter and the number of

Xiang Ding; Lian-Yue Yang; Gen-Wen Huang; Wei Wang; Wei-Qun Lu

2004-01-01

363

Effects of Ginseng and Echinacea on Cytokine mRNA Expression in Rats  

PubMed Central

The aim of the study was to determine the effect of ginseng and echinacea on the mRNA expression of IL-10, TNF-?, and TGF-?1 in healthy rats. Six-week-old male Fischer 344 rats (n = 48) were used. The animals were divided into three equal groups, as follows: control (C); ginseng (G); echinacea (E). While the C group was fed a standard rat diet (Purina) ad libitum for a period of 40 days, the G and E groups animals received the same diet containing 0.5?g/kg of Panax ginseng root powder and 0.75?g/kg of Echinacea purpurea root powder, respectively. Blood samples were obtained from 8 rats in each group after 20 and 40 days of treatment, and the mRNA expression of IL-10, TNF-?, and TGF-?1 was determined. After 20 days of treatment, the expression of IL-10 mRNA in the G group was different from the C group (P < 0.05); however, after 40 days of treatment, there was no difference between the groups. There was no difference after 20 and 40 days of treatment between the groups with respect to the expression of TGF-?1 mRNA. After 20 days of treatment, the expression of TNF-? mRNA in the E group was higher (P < 0.05) than the C group. After 40 days of treatment, the expression of TNF-? mRNA was similar in all of the groups. Based on the current study, the increase in expression of IL-10 mRNA in the G group and the increase in expression of TNF-? mRNA in the E group support the use of these plants for purposes of modulating the immune system. However, a more detailed study regarding the effects of ginseng and echinacea on these cytokines and other cytokines is needed. PMID:22666172

Ulu???k, Deniz; Keskin, Ercan

2012-01-01

364

Quality control of mRNA 3'-end processing is linked to the nuclear exosome  

Microsoft Academic Search

An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control. Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm. In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA

Patricia Hilleren; Terri McCarthy; Michael Rosbash; Roy Parker; Torben Heick Jensen

2001-01-01

365

mRNA expression analysis of the physiological responses to ammonia infusion in rainbow trout.  

PubMed

We recently reported that tissue levels of Rhesus (Rh) mRNA in rainbow trout changed in response to high-external ammonia (HEA). To investigate whether or not these changes could be due to elevated plasma ammonia levels, we infused rainbow trout for 12 h with 140 mmol L(-1) NH(4)HCO(3), or with 140 mmol L(-1) NaCl as a control for the effects of infusion. We also analyzed the effects of dorsal aortic catheterization alone, without infusion. Catheterization alone resulted in an elevated ammonia excretion rate, a downregulation of Rhbg mRNA in the brain, and mRNA upregulations of Rhbg, Rhcg1, and Rhcg2 in the gill, Rhbg and Rhcg1 in the skin, and Rhag in the erythrocytes. In NH(4)HCO(3)-infused fish, plasma cortisol peaked at 6 h, erythrocyte Rhag mRNA was downregulated, gill Rhbg, Rhcg1, and Rhcg2 mRNA were upregulated, and skin Rhbg mRNA was also upregulated. NaCl infusion resulted in elevated plasma ammonia and ammonia excretion rates as well as gill mRNA upregulations of Rhbg, carbonic anhydrase, NHE2, H(+)-ATPase, Na(+)/K(+)-ATPase. Taken together, the results indicated that infusion of NH(4)HCO(3) induced a similar pattern of Rh transcript changes as that seen when fish were exposed to HEA. Second, catheterization alone, as well as isotonic NaCl infusion, significantly altered mRNA levels, highlighting the necessity for careful data interpretation and inclusion of appropriate controls for gene expression studies in fish that have undergone anaesthesia/surgery and infusion procedures. Finally, elevated plasma ammonia and cortisol may both be involved in the signaling mechanism for Rh gene regulation. PMID:19377886

Nawata, C Michele; Wood, Chris M

2009-10-01

366

iBioSeminar: The Life of Eukaryotic mRNA  

NSDL National Science Digital Library

The control of mRNA production and function is a key aspect of the regulation of gene expression. Eukaryotic cells, the control of mRNA localization, translation and degradation in the cytoplasm allow for the proper regulation of the amount, duration, and location of protein production. The basic mechanisms of these processes are understood and reveal that the mechanisms of localization, translation, and degradation are interconnected.

Roy Parker (University of Arizona and Howard Hughes Medical Institute; )

2010-10-07

367

Efficient genetic modification of murine dendritic cells by electroporation with mRNA  

Microsoft Academic Search

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti–tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient

Sonja Van Meirvenne; Lieven Straetman; Carlo Heirman; Melissa Dullaers; Catherine De Greef; Viggo Van Tendeloo; Kris Thielemans

2002-01-01

368

Estrogen Receptor ? mRNA in Colon Cancer Cells: Growth Effects of Estrogen and Genistein  

Microsoft Academic Search

Knowledge regarding the expression of the recently cloned estrogen receptor ? (ER?) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ER? mRNA, but lacked ER? mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while

Naoya Arai; Anders Ström; Joseph J. Rafter; Jan-Åke Gustafsson

2000-01-01

369

The Xenopus ELAV Protein ElrB Represses Vg1 mRNA Translation during Oogenesis  

Microsoft Academic Search

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3- untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and

Lucy J. Colegrove-Otero; Agathe Devaux; Nancy Standart

2005-01-01

370

Moisturizers change the mRNA expression of enzymes synthesizing skin barrier lipids  

Microsoft Academic Search

In a previous study, 7-week treatment of normal human skin with two test moisturizers, Complex cream and Hydrocarbon cream,\\u000a was shown to affect mRNA expression of certain genes involved in keratinocyte differentiation. Moreover, the treatment altered\\u000a transepidermal water loss (TEWL) in opposite directions. In the present study, the mRNA expression of genes important for\\u000a formation of barrier lipids, i.e., cholesterol,

Izabela Buraczewska; Berit Berne; Magnus Lindberg; Marie Lodén; Hans Törmä

2009-01-01

371

Molecular Characterization of Fetal Alcohol Syndrome Using mRNA Differential Display  

Microsoft Academic Search

The molecular pathogenesis of fetal alcohol syndrome (FAS) has not been well elucidated. The technique of mRNA differential display was used to characterize the etiology and to identify potential markers for FAS. Out of approximately 1,080 mRNA transcripts in mouse embryos that were analyzed, the levels of three mRNAs were altered by ethanol. Two of these mRNAs (one novel and

Insong James Lee; Yunjo Soh; Byoung Joon Song

1997-01-01

372

CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.  

PubMed

The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (?-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ?50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest. PMID:24603867

Zebec, Ziga; Manica, Andrea; Zhang, Jing; White, Malcolm F; Schleper, Christa

2014-04-01

373

Dynactin suppresses the retrograde movement of apically localized mRNA in Drosophila blastoderm embryos.  

PubMed

Motor dependent transport of mRNA is a key mechanism in axis specification during development. Apical transport and anchoring of wingless and pair-rule transcripts in the Drosophila syncytial blastoderm embryo is mediated by cytoplasmic Dynein, the major minus end directed microtubule dependent molecular motor. Here, we show that, despite apical transport of mRNA being highly directional, mRNA particles often pause and move backward toward the plus ends of microtubules. We suggest that this retrograde movement helps overcome cellular obstructions. We show that the plus end movement of apical mRNA is independent of the major plus end microtubule motors Kinesin-1 and Kinesin-2. In contrast, Dynactin, a Dynein processivity factor, is required to suppress retrograde mRNA movements, as well as for efficient minus end motility. We propose that Dynein itself, rather than the activity of a plus end motor, is responsible for the plus end movements of the mRNA and that Dynactin is involved in preventing short reverse movements of the Dynein motor, known to occur in vitro. PMID:17901156

Vendra, Georgia; Hamilton, Russell S; Davis, Ilan

2007-11-01

374

Erythroid cell-specific determinants of alpha-globin mRNA stability.  

PubMed Central

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells. Images PMID:7969150

Weiss, I M; Liebhaber, S A

1994-01-01

375

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures  

PubMed Central

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

376

Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex  

PubMed Central

The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms. PMID:25393401

Schnell, Steven J.; Ma, Jiong; Yang, Weidong

2014-01-01

377

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis  

PubMed Central

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near ? duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near ? duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

378

Paramyxovirus mRNA editing, the "rule of six" and error catastrophe: a hypothesis.  

PubMed

The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe. PMID:15958664

Kolakofsky, Daniel; Roux, Laurent; Garcin, Dominique; Ruigrok, Rob W H

2005-07-01

379

Viscum album-Mediated COX-2 Inhibition Implicates Destabilization of COX-2 mRNA  

PubMed Central

Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1?-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA. PMID:25664986

Saha, Chaitrali; Hegde, Pushpa; Friboulet, Alain; Bayry, Jagadeesh; Kaveri, Srinivas V.

2015-01-01

380

Gravitational loading of a simulated launch alters mRNA expression in osteoblasts  

NASA Technical Reports Server (NTRS)

Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

Fitzgerald, J.; Hughes-Fulford, M.

1996-01-01

381

High level of p37?-mRNA relative to p110?-mRNA in neuroblastoma tumors correlates with poor patient survival.  

PubMed

Alterations in the PI3K/Akt pathway, a pathway that promotes proliferation and oncogenic transformation, are common in various cancers. In neuroblastoma, activation of Akt is correlated with aggressive disease although mutations in genes of this pathway are rare. Previous findings include a few mutations in PIK3CD, the gene encoding PI3K catalytic subunit delta, p110?. We recently reported that an alternatively spliced form of p110?, called p37?, had cell proliferative properties and was over-expressed in ovarian and colorectal tumors. Here, we investigated p37? in neuroblastoma primary tumors of different stages using qPCR (TaqMan) for gene expression analysis (46 samples) and Western blot for protein analysis (22 samples). Elevated levels of both p37?-mRNA and p110?-mRNA were detected in metastasizing neuroblastoma tumors compared to normal adrenal gland (P < 0.05), and higher expression of p37?-mRNA relative to p110?-mRNA in neuroblastoma non-survivor patients compared to survivors (P < 0.01). p37?-Protein levels but not p110? levels correlated with increased pAKT(T308) and pERK levels. The p37?-mRNA levels did not correlate with the protein levels, indicating major regulation at the translational/protein level. Deregulation of signaling pathways is a hallmark of cancer development. Here, we show that p37?, a kinase-dead isoform of the PI3K catalytic subunit p110?, is over-expressed in neuroblastoma tumors, and that it correlates with the activation of both PI3K/Akt- and RAS-signaling pathways. PMID:24026661

Fransson, Susanne; Ejeskär, Katarina

2013-12-01

382

The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage  

PubMed Central

Aconitase is an iron–sulfur protein and a major enzyme of the TCA cycle that catalyzes the conversion of citrate to isocitrate under iron-rich conditions. In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3?UTR and stabilize it when intracellular iron become scarce. The small regulatory RNA (sRNA) RyhB has previously been shown to promote RNase E-dependent degradation of acnB mRNA when it was expressed from an ectopic arabinose-dependent promoter, independently of intracellular iron levels. In marked contrast, we report here that expression of RyhB under low-iron conditions did not result in acnB mRNA degradation even when RyhB was bound to acnB ribosome binding site (RBS). Genetic and biochemical evidence suggested that, under low-iron conditions, apo-AcnB bound to acnB 3?UTR close to a RNase E cleavage site that is essential for RyhB-induced acnB mRNA degradation. Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site. This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability. PMID:25092924

Benjamin, Julie-Anna M.; Massé, Eric

2014-01-01

383

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines  

SciTech Connect

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER?ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER? and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor ? (ER?) positive (ER+) breast cancer cells compared to ER? cells. However, the presence of LRH-1 protein in ER? cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER? breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER? compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER? versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ER?. Our data demonstrates that in ER? cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER? cells as well as ER? tumors suggests a possible role in the development of ER? tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER? and ER+ breast cancer.

Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia) [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia)] [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia) [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia)] [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

2013-08-30

384

Prostaglandin E2 increases proenkephalin mRNA level in rat astrocyte-enriched culture.  

PubMed

The effect of prostaglandin E2 (PGE2) on proenkephalin (proENK) mRNA expression in primary cultured rat astrocytes was studied. The proENK mRNA level was significantly increased about 3.3-fold 4 h after PGE2 (10 microM) treatment and this increase was potentiated by the pre-treatment with cycloheximide (CHX; 15 microM) about 1.7-fold as much as PGE2 alone treated cells. The pretreatment with staurosporine (1 microM) completely inhibited the increase of PGE2-induced proENK mRNA level, although only a partial inhibition of PGE2-induced proENK mRNA level (approximately 1.5-fold) by H89 (10 microM) was observed. The increase of PGE2-induced proENK mRNA level was not affected by the pretreatment with PD98059 (1, 5, and 10 microM), omega-conotoxin GIVA (1 microM), nimodipine (1 microM), calmidazolium (1 microM), or KN-62 (1 microM). In addition to the proENK mRNA level, PGE2 also increased c-Fos (approximately 4.3-fold), Fra-1 ( approximately 3.8 fold), and Fra-2 (approximately 8.2-fold) protein levels at 4 h after drug treatment. However, c-Jun, JunB, and JunD protein levels were not affected by PGE2. Indeed, PGE2 failed to up-regulate c-jun mRNA expression as well as its protein product. Surprisingly, although three Jun proteins were not induced by PGE2, AP-1 and ENKCRE-2 DNA binding activities were increased by PGE2, (approximately 5 and approximately 2.8-fold, respectively) and which were effectively reduced by CHX (approximately 2.5 and 2-fold, respectively). In western blot analyses, PGE2 enhanced the phosphorylation of CREB (approximately 2.6-fold at 1 h), and CHX showed a potentiative effect on PGE2-induced CREB phosphorylation ( approximately 1.7 fold at 1 h) which is similar to the action on proENK mRNA regulation. Our results suggest that PGE2 increases proENK mRNA expression via activating serine/threonine protein kinase such as PKA, but not calcium/calmodulin dependent protein kinase and MAPK. In addition, phosphorylation of CREB rather than the increase of AP-1 may have a possible role at least early stage in PGE2-induced proENK mRNA level and CHX-evoked potentiation. PMID:9757037

Won, J S; Suh, H W; Kim, Y H; Song, D K; Huh, S O; Lee, J K; Lee, K J

1998-10-01

385

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.  

PubMed

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration. PMID:16248107

Worch, Remigiusz; Stepinski, Janusz; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Mazza, Catherine; Cusack, Stephen; Stolarski, Ryszard; Darzynkiewicz, Edward

2005-01-01

386

The Critical Role of mRNA Destabilizing Protein Heterogeneous Nuclear Ribonucleoprotein D in 3? Untranslated Region–Mediated Decay of Low-Density Lipoprotein Receptor mRNA in Liver Tissue  

PubMed Central

Objective Previous studies showed that low-density lipoprotein receptor (LDLR) mRNA 3? untranslated region (UTR) contains regulatory elements responsible for rapid mRNA turnover in hepatic cells and mediates the mRNA stabilization induced by berberine (BBR). Here, we elucidate the underlying mechanism of BBR’s action by characterizing mRNA-binding proteins that modulate LDLR mRNA decay via 3? UTR in liver tissue in vivo. Approach and Results We generated a transgenic mouse model (Alb-Luc-UTR) that expresses Luc-LDLR3? UTR reporter gene driven by the albumin promoter to study 3? UTR function in mediating LDLR mRNA decay in liver tissue. We show that treating Alb-Luc-UTR mice with BBR led to significant increases in hepatic bioluminescence signals, Luc-UTR mRNA, and LDLR mRNA levels as compared with control mice. These effects were accompanied by specific reductions of mRNA decay-promoting factor heterogeneous nuclear ribonucleoprotein D (hnRNP D) in liver of BBR-treated mice. Knockdown and overexpression studies further demonstrated that hnRNP D p37 isoform plays a major role in promoting hepatic LDLR mRNA degradation. In addition, we examined LDLR mRNA half-life, Luc-UTR reporter activity, and hnRNP D expression levels in cell lines derived from extrahepatic tissues. We demonstrated that strengths of 3? UTR in promoting mRNA degradation correlate with hnRNP D cellular abundances in nonhepatic cell lines, thereby suggesting its involvement in LDLR mRNA degradation beyond liver tissue. Conclusions hnRNP D is critically involved in LDLR mRNA degradation in liver tissue in vivo. The inverse relationship of hnRNP D abundance with LDLR mRNA levels after BBR treatment suggests the potential of hnRNP D of being a novel therapeutic target for LDL cholesterol lowering. PMID:24158514

Singh, Amar Bahadur; Li, Hai; Kan, Chin Fung Kelvin; Dong, Bin; Nicolls, Mark R.; Liu, Jingwen

2014-01-01

387

Stability of mRNA influences osteoporotic bone mass via CNOT3  

PubMed Central

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-?B ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-?B (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3?-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3?-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis. PMID:24550297

Watanabe, Chiho; Morita, Masahiro; Hayata, Tadayoshi; Nakamoto, Tetsuya; Kikuguchi, Chisato; Li, Xue; Kobayashi, Yasuhiro; Takahashi, Naoyuki; Notomi, Takuya; Moriyama, Keiji; Yamamoto, Tadashi; Ezura, Yoichi; Noda, Masaki

2014-01-01

388

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding  

PubMed Central

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5?-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5?-UTR. PMID:10809669

Vytvytska, Oresta; Moll, Isabella; Kaberdin, Vladimir R.; von Gabain, Alexander; Bläsi, Udo

2000-01-01

389

mRNA stability as a function of striated muscle oxidative capacity.  

PubMed

A change in mRNA stability alters the abundance of mRNA available for translation and is emerging as a critical pathway influencing gene expression. Variations in the stability of functional and regulatory mitochondrial proteins may contribute to the divergent mitochondrial densities observed in striated muscle. Thus we hypothesized that the stability of mRNAs encoding for regulatory nuclear and mitochondrial transcription factors would be inversely proportional to muscle oxidative capacity and would be facilitated by the activity of RNA binding proteins (RBPs). The stability of mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1? (PGC-1?), and nuclear respiratory factor 2? (NRF-2?) mRNA was assessed in striated muscles with distinct oxidative capacities using in vitro decay assays. All three mitochondrial regulators were rapidly degraded in cardiac and slow-twitch red (STR) muscle, resulting in a ?60-65% lower (P < 0.05) mRNA half-life (t(1/2)) compared with fast-twitch white (FTW) fibers. This accelerated rate of Tfam mRNA decay was matched by a 2.5-fold increase in Tfam transcription in slow- compared with fast-twitch muscle (P = 0.05). Protein expression of four unique RBPs [AU-rich binding factor 1 (AUF1), human antigen R (HuR), KH-homology splicing regulatory protein (KSRP), and CUG binding protein 1 (CUGBP1)] believed to modulate mRNA stability was elevated in cardiac and STR muscles (P < 0.05) and was moderately associated with the decay of Tfam, PGC-1?, and NRF-2? mRNA. Variable rates of transcript degradation were apparent when comparing all transcripts within the same muscle type. Thus the distribution of RBPs appears to follow a fiber-type specific pattern and subsequently functions to alter the stability of specific mitochondrial regulators in a transcript- and tissue-specific fashion. PMID:22718808

D'souza, Donna; Lai, Ruanne Y J; Shuen, Michael; Hood, David A

2012-08-15

390

UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

Dalgaard, Louise T., E-mail: ltd@ruc.dk [Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Department of Science, Systems and Models, Roskilde University (Denmark)

2012-01-06

391

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes  

PubMed Central

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1. PMID:19657054

Peffers, Mandy J.; McKay, Tristan R.; Lowe, Emma T.; Khan, Wasim S.; Hardingham, Timothy E.; Clegg, Peter D.

2009-01-01

392

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.  

PubMed

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1. PMID:19657054

Tew, Simon R; Peffers, Mandy J; McKay, Tristan R; Lowe, Emma T; Khan, Wasim S; Hardingham, Timothy E; Clegg, Peter D

2009-10-01

393

Maternal mRNA expression levels of H19 are inversely associated with risk of macrosomia  

PubMed Central

Introduction To investigate the associations between the mRNA levels of H19 in term placenta and risk of macrosomia. Material and methods Term placentas were collected from 37 macrosomia and 37 matched neonates with normal birth weight (controls) born in Changzhou Women and Children Health Hospital, Jiangsu province, P. R. China from March 1 to June 30, 2008. The mRNA levels of H19 in those placentas were measured by real-time polymerase chain reaction (PCR). Simple and multiple logistic regression models were used to explore the risk factors in the development of macrosomia. All analyses were performed using Stata 10.0 (StataCorp, College Station, Texas, USA). Results The average H19 mRNA level of the macrosomia group was 1.450 ±0.456 while in the control group it was 2.080 ±1.296. Based on the result of Student's t test, there was a significant difference in H19 mRNA level between the macrosomia group and the control group (p = 0.008). After controlling for potential confounders, the multivariable adjusted odds ratio (OR) of macrosomia for those in the highest tertile of H19 mRNA level was 0.12 (95% CI: 0.02–0.59) when compared to those in the lowest tertile (p for linear trend = 0.009). Conclusions The term placental H19 mRNA levels were inversely related to the occurrence of macrosomia. Our findings suggest that the low expression of H19 mRNA may contribute to the development of macrosomia. PMID:25097584

Jiang, Hua; Yu, Yang; Xun, Pengcheng; Zhang, Jun; Luo, Guanghua

2014-01-01

394

Conservative sequences in 3'UTR of TCRzeta mRNA regulate TCRzeta in SLE T cells.  

PubMed

We have demonstrated that T-cell receptor zeta (zeta) mRNA with a 562-bp deleted alternatively spliced 3'-untranslated region (3'UTR) observed in T cells of patients with systemic lupus erythematosus (SLE) can lead to a reduction in zeta and TCR/CD3 (J. Immunol., 2003 & 2005). To determine the region in zeta mRNA 3'UTR for the regulation of zeta, zeta mRNA with 3'UTR truncations ligated into pDON-AI was used to infect murine T-cell hybridoma MA5.8 cells, which do not contain zeta. As a Western blot analysis demonstrated the importance of the regions from +871 to +950, containing conservative sequence 1 (CS1), and +1070 to +1136, containing CS2, for the production of zeta, we constructed MA5.8 mutants carrying zeta mRNA 3'UTR with deletions of these regions (DeltaCS1 and DeltaCS2 mutants). Western blot and FACS analyses showed significant reduction in the cell surface zeta and TCR/CD3 in both these mutants, and IL-2 production was decreased, compared with MA5.8 cells transfected with wild-type zeta mRNA. Furthermore, real-time PCR demonstrated the instability of zeta mRNA with 3'UTR deletions in these MA5.8 mutants. In conclusion, CS1 and CS2 may be responsible for the regulation of zeta and TCR/CD3 through the stability of zeta mRNA in SLE T cells. PMID:18177736

Tsuzaka, Kensei; Itami, Yuka; Kumazawa, Chika; Suzuki, Miyuki; Setoyama, Yumiko; Yoshimoto, Keiko; Suzuki, Katsuya; Abe, Tohru; Takeuchi, Tsutomu

2008-03-01

395

Chapter 1. Methods to study no-go mRNA decay in Saccharomyces cerevisiae.  

PubMed

In eukaryotic cells, conserved mRNA surveillance systems target and degrade aberrant mRNAs, eliminating translation errors that occur during protein synthesis and thereby imposing quality control of gene expression. Two such cytoplasmic quality control systems, nonsense-mediated mRNA decay and nonstop mRNA decay, have evolved to target mRNAs with aberrancies in translation. A third novel quality control system has been identified for yeast mRNAs with defects in translation elongation due to strong translation pause sites. This subset of mRNAs with ribosome pause sites is recognized and targeted for degradation by an endonucleolytic cleavage in a process referred to as no-go mRNA decay (NGD). The methods described herein are designed to aid in the study of NGD in Saccharomyces cerevisiae. They include procedures to create an efficient translation elongation pause, assay decay characteristics of NGD substrates, and characterize NGD-dependent endonucleolytic cleavage of mRNA. The logic of the design and methods described can be modulated and used for the identification and analysis of novel RNA quality control pathways in other organisms. PMID:19215751

Doma, Meenakshi K

2008-01-01

396

GLUT3 protein and mRNA in autopsy muscle specimens  

NASA Technical Reports Server (NTRS)

GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

Stuart, C. A.; Wen, G.; Jiang, J.

1999-01-01

397

The Streptococcus mutans irvA gene encodes a trans-acting riboregulatory mRNA.  

PubMed

In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation. PMID:25574948

Liu, Nan; Niu, Guoqing; Xie, Zhoujie; Chen, Zhiyun; Itzek, Andreas; Kreth, Jens; Gillaspy, Allison; Zeng, Lin; Burne, Robert; Qi, Fengxia; Merritt, Justin

2015-01-01

398

Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review)  

PubMed Central

Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5? or 3? to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3?-untranslated (3?-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs. PMID:24452120

DI LIEGRO, CARLO MARIA; SCHIERA, GABRIELLA; DI LIEGRO, ITALIA

2014-01-01

399

Kinetic models of the interference of gene transcription to ncRNA and mRNA  

NASA Astrophysics Data System (ADS)

The experiments indicate that the transcription of genes into ncRNA can positively or negatively interfere with transcription into mRNA. We propose two kinetic models describing this effect. The first model is focused on the ncRNA-induced chromatin modification facilitating the transcription of the downstream gene into mRNA. The second model includes the competition between the transcription into ncRNA and the binding of activator to a regulatory site of the downstream gene transcribed into mRNA. Our analysis based on the mean-field kinetic equations and Monte Carlo simulations shows the likely dependences of the transcription rate on RNA polymerase concentration in situations with different rate-limiting steps. Our models can also be used to scrutinize the dependence of the transcription rate on other kinetic parameters. Our kinetic Monte Carlo simulations show that the first model predicts stochastic bursts in the mRNA formation provided that the transcription into ncRNA is slow, while the second model predicts in addition anti-phase stochastic bursts in the mRNA and ncRNA formation provided that that the protein attachment to and detachment from a regulatory site is slow.

Zhdanov, Vladimir P.

2011-06-01

400

Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway  

PubMed Central

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5? end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs. PMID:23427269

Quaresma, Alexandre Jose Christino; Sievert, Rachel; Nickerson, Jeffrey A.

2013-01-01

401

Cytochrome P450IA mRNA expression in feral Hudson River tomcod.  

PubMed

We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers. PMID:1855491

Kreamer, G L; Squibb, K; Gioeli, D; Garte, S J; Wirgin, I

1991-06-01

402

Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review).  

PubMed

Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5' or 3' to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3'-untranslated (3'-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs. PMID:24452120

Di Liegro, Carlo Maria; Schiera, Gabriella; Di Liegro, Italia

2014-04-01

403

Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA  

SciTech Connect

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

1994-12-31

404

[Purification and identification of mRNA of immune (gamma) interferon from human splenocytes].  

PubMed

The isolation of total RNA from primary culture of human splenocytes and its physico-chemical and biological properties are described. Human splenocytes are characterized by a high content of mRNA of human immune interferon, low content of total RNA and an extremely high activity of RNAases. Therefore it was necessary to elaborate conditions for the isolation of mRNA without DNA contaminants in the presence of extensive inhibitors of the RNAase activity. These include cell homogenization, separation of cytoplasm at -10 degrees C and treatment by RNAase inhibitors--ribonucleoside-vanadyl complexes or a combination of aurin-tricarboxylic acid with dithiothreitol. The resulting preparations of total RNA were purified by chromatography on oligo (dT)-cellulose and translated in a cell-free system from rabbit reticulocytes. These preparations were free of nonspecific translation inhibitors which are normally present in the lymphoid cells mRNA. In a cell-free system mRNA of human splenocytes induced with staphylococcal enterotoxin A code the synthesis of biologically active interferon which was identified as immune (gamma) human interferon, using a serological analysis. The preparations of immune interferon mRNA obtained under the conditions described above can further be used for cloning of the corresponding gene in bacterial cells. PMID:6416307

Liakh, L A; Khil'ko, S N; Aspetov, R D; Nosik, D N; Novokhatski?, A S

1983-10-01

405

Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus  

SciTech Connect

Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.

1987-10-01

406

Genome-wide computational identification of bicistronic mRNA in humans.  

PubMed

Mammalian bicistronic mRNA is a recently discovered mammalian gene structure. Several reported cases of mammalian bicistronic mRNA indicated that genes of this structure play roles in some important biological processes. However, a genome-wide computational identification of bicistronic mRNA in mammalian genome, such as human genome, is still lacking. Here we used a comparative genomics approach to identify the frequency of human bicistronic mRNA. We then validated the result by using a new support vector machine (SVM) model. We identified 43 human bicistronic mRNAs in 30 distinct genes. Our literature analysis shows that our method recovered 100 % (6/6) of the previously known bicistronic mRNAs which had been experimentally confirmed by other groups. Our graph theory-based analysis and GO analysis indicated that human bicistronic mRNAs are prone to produce different yet closely functionally related proteins. In addition, we also described and analyzed three different mechanisms of ORF fusion. Our method of identifying bicistronic mRNAs in human genome provides a model for the computational identification of characteristic gene structures in mammalian genomes. We anticipate that our data will facilitate further molecular characterization and functional study of human bicistronic mRNA. PMID:22945903

Lu, Yiming; Zhang, Yanchun; Hang, Xingyi; Qu, Wubin; Lubec, Gert; Chen, Changsheng; Zhang, Chenggang

2013-02-01

407

Stochastic theory of protein synthesis and polysome: ribosome profile on a single mRNA transcript  

E-print Network

The process of polymerizing a protein by a ribosome, using a messenger RNA (mRNA) as the corresponding template, is called {\\it translation}. Ribosome may be regarded as a molecular motor for which the mRNA template serves also as the track. Often several ribosomes may translate the same (mRNA) simultaneously. The ribosomes bound simultaneously to a single mRNA transcript are the members of a polyribosome (or, simply, {\\it polysome}). Experimentally measured {\\it polysome profile} gives the distribution of polysome {\\it sizes}. Recently a breakthrough in determining the instantaneous {\\it positions} of the ribosomes on a given mRNA track has been achieved and the technique is called {\\it ribosome profiling} \\cite{ingolia10,guo10}. Motivated by the success of these techniques, we have studied the spatio-temporal organization of ribosomes by extending a theoretical model that we have reported elsewhere \\cite{sharma11}. This extended version of our model incorporates not only (i) mechano-chemical cycle of individual ribomes, and (ii) their steric interactions, but also (iii) the effects of (a) kinetic proofreading, (b) translational infidelity, (c) ribosome recycling, and (d) sequence inhomogeneities. The theoretical framework developed here will serve in guiding further experiments and in analyzing the data to gain deep insight into various kinetic processes involved in translation.

Ajeet K. Sharma; Debashish Chowdhury

2011-08-18

408

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma.  

PubMed

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level. PMID:2894088

Höfler, H; Pütz, B; Ruhri, C; Wirnsberger, G; Klimpfinger, M; Smolle, J

1987-01-01

409

Identification of a cis-acting element that localizes mRNA to synapses  

PubMed Central

Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cis-acting RNA elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3? UTR is sufficient for export into distal neurites, whereas the 5? UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5? UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process. PMID:22383561

Meer, Elliott J.; Wang, Dan Ohtan; Kim, Sangmok; Barr, Ian; Guo, Feng; Martin, Kelsey C.

2012-01-01

410

Integrated miRNA and mRNA expression profiling to identify mRNA targets of dysregulated miRNAs in non-obstructive azoospermia  

PubMed Central

The aim of this study was to identify mRNA targets of dysregulated miRNAs through the integrated analysis of miRNA and mRNA expression profiling in men with normal versus impaired spermatogenesis. The expression of mRNAs and miRNAs in testicular tissues obtained from males with non-obstructive azoospermia (NOA, n = 4) or obstructive azoospermia (OA, n = 3) with normal spermatogenesis was analyzed using microarray technology. Some of the most interesting results were validated by real time PCR using samples from the same cohort. Ninety-three miRNAs and 4172 mRNAs were differentially expressed in the NOA and normozoospermic OA patients. In addition to confirming that significantly dysregulated genes and miRNAs play pivotal roles in NOA, promising correlation signatures of these miRNA/mRNA pairs were discovered in this study. The functional classification of the miRNA/mRNA pairs revealed that differentially expressed genes