Sample records for chemoattractant protein-1 expression

  1. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp; Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510; Yoshizaki, Takayuki

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytesmore » by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.« less

  2. Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

    PubMed

    Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

    1998-01-01

    These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

  3. Ovarian kisspeptin expression is related to age and to monocyte chemoattractant protein-1.

    PubMed

    Merhi, Zaher; Thornton, Kimberley; Bonney, Elizabeth; Cipolla, Marilyn J; Charron, Maureen J; Buyuk, Erkan

    2016-04-01

    The objective of this study was to test the hypothesis that ovarian kisspeptin (kiss1) and its receptor (kiss1r) expression are affected by age, obesity, and the age- and obesity-related chemokine monocyte chemoattractant protein-1 (MCP-1). Ovaries from reproductive-aged and older C57BL/6J mice fed normal chow (NC) or high-fat (HF) diet, ovaries from age-matched young MCP-1 knockout and young control mice on NC, and finally, cumulus and mural granulosa cells (GCs) from women who underwent in vitro fertilization (IVF) were collected. Kiss1, kiss1r, anti-Mullerian hormone (AMH), and AMH receptor (AMHR-II) messenger RNA (mRNA) expression levels were quantified using real-time polymerase chain reaction (RT-PCR). In mouse ovaries, kiss1 and kiss1r mRNA levels were significantly higher in old compared to reproductive-aged mice, and diet-induced obesity did not alter kiss1 or kiss1r mRNA levels. Compared to young control mice, young MCP-1 knockout mice had significantly lower ovarian kiss1 mRNA but significantly higher AMH and AMHR-II mRNA levels. In human cumulus GCs, kiss1r mRNA levels were positively correlated with age but not with BMI. There was no expression of kiss1 mRNA in either cumulus or mural GCs. These data suggest a possible age-related physiologic role for the kisspeptinergic system in ovarian physiology. Additionally, the inflammatory MCP-1 may be associated with kiss1 and AMH genes, which are important in ovulation and folliculogenesis, respectively.

  4. Effects of Coriolus versicolor polysaccharide B on monocyte chemoattractant protein 1 gene expression in rat.

    PubMed

    Song, Lie-Chang; Chen, Hai-Sheng; Lou, Ning; Song, Chang; Zeng, Jun; Fu, Ting-Huan

    2002-06-01

    To investigate the effect of Coriolus versicolor polysaccharide B (CVPS-B), a new water-soluble component of polysaccharides from the fungus Coriolus versicolor (Fr) L on monocyte chemoattractant protein-1 (MCP-1) gene expression in rat splenocytes. Expression of MCP-1 mRNA in rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR) with beta- actin as an internal standard. Sequencing of RT-PCR products was performed to confirm their specificity in MCP-1 gene composition. (1) Without pre-treatment of lipopolysaccharide (LPS), the relative MCP-1 mRNA expression ratios (MCP-1/beta-actin) for the saline control group and for CVPS-B groups in 3 different doses (10, 20, and 30 mg . kg-1 . d-1, ip, for 4 d) were 1.4 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.5, and 1.5 +/- 0.4, respectively (P > 0.05); (2) LPS (10 microg . kg-1, ip) enhanced the expression of MPC-1 mRNA by the ratio of 114 %; (3) pre-treatment with CVPS-B of 4 different doses (5, 10, 30, and 50 mg . kg-1 . d-1, ip, for 4 d) decreased the LPS induced expression of MPC-1 mRNA by the ratios of 51 %, 70 %, 84 %, and 99 %, respectively (n = 6). In a dose-related fashion, CVPS-B inhibited the expression of MCP-1 mRNA induced by LPS in the rat splenocytes, but did not significantly affect the expression of MPC-1 mRNA in the normal rat.

  5. Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury.

    PubMed

    Jocks, T; Zahner, G; Freudenberg, J; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

    1996-06-01

    To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly enhanced compared with controls. In the isolated perfused kidney, antibody and complement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody and complement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries.

  6. Monocyte Chemoattractant Protein 1 (MCP-1) in Obesity and Diabetes

    PubMed Central

    Panee, Jun

    2012-01-01

    Monocyte chemoattractant protein-1 (MCP-1) is the first discovered and most extensively studied CC chemokine, and the amount of studies on its role in the etiologies of obesity- and diabetes-related diseases have increased exponentially during the past 2 decades. This review attempted to provide a panoramic perspective of the history, regulatory mechanisms, functions, and therapeutic strategies of this chemokine. The highlights of this review include the roles of MCP-1 in the development of obesity, diabetes, cardiovascular diseases, insulitis, diabetic nephropathy, and diabetic retinopathy. Therapies that specifically or non-specifically inhibit MCP-1 overproduction have been summarized. PMID:22766373

  7. Alterations of peroxisome proliferator-activated receptor γ and monocyte chemoattractant protein 1 gene expression in the nitrofen-induced hypoplastic lung.

    PubMed

    Gosemann, Jan-Hendrik; Doi, Takashi; Kutasy, Balazs; Friedmacher, Florian; Dingemann, Jens; Puri, Prem

    2012-05-01

    Peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in normal lung development. Peroxisome proliferator-activated receptor γ messenger RNA (mRNA) is detectable at 18 days of gestation in fetal rat lungs, and levels peak just before birth. Peroxisome proliferator-activated receptor γ agonists are reported to stimulate lung development, whereas inhibition of PPARγ disrupts postnatal lung maturation. Monocyte chemoattractant protein 1 (MCP-1), which is inhibited by PPARγ, is reported to disrupt late lung morphogenesis. This study was designed to investigate the hypothesis that PPARγ expression is downregulated and that MCP-1 expression is upregulated during the late stages of lung development in nitrofen-induced hypoplastic lungs. Pregnant rats were treated with nitrofen or vehicle on D9. RNA was extracted from fetal lungs (D18 and D21), and relative mRNA expression levels of PPARγ and MCP-1 were determined by reverse transcriptase-polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression/distribution of PPARγ and MCP-1. Relative mRNA expression levels of PPARγ were significantly downregulated in the nitrofen group compared with controls on D21, whereas MCP-1 levels were upregulated. Immunohistochemical study showed markedly decreased PPARγ and increased MCP-1 immunoreactivity in the nitrofen-induced hypoplastic lungs compared with controls on gestational day 21. Altered pulmonary gene expression of PPARγ and MCP-1 during late gestation may impair lung development and maturation, contributing to pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia model. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Cyclooxygenase metabolites mediate glomerular monocyte chemoattractant protein-1 formation and monocyte recruitment in experimental glomerulonephritis.

    PubMed

    Schneider, A; Harendza, S; Zahner, G; Jocks, T; Wenzel, U; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

    1999-02-01

    Monocyte chemoattractant protein-1 (MCP-1) has been shown to play a significant role in the recruitment of monocytes/macrophages in experimental glomerulonephritis. Whereas a number of inflammatory mediators have been characterized that are involved in the expression of MCP-1 in renal disease, little is known about repressors of chemokine formation in vivo. We hypothesized that cyclooxygenase (COX) products influence the formation of MCP-1 and affect inflammatory cell recruitment in glomerulonephritis. The effect of COX inhibitors was evaluated in the antithymocyte antibody model and an anti-glomerular basement membrane model of glomerulonephritis. Rats were treated with the COX-1/COX-2 inhibitor indomethacin and the selective COX-2 inhibitors meloxicam and SC 58125. Animals were studied at 1 hour, 24 hours, and 5 days after induction of the disease. Indomethacin, to a lesser degree the selective COX-2 inhibitors, enhanced glomerular MCP-1 and RANTES mRNA levels. Indomethacin enhanced glomerular monocyte chemoattractant activity an the infiltration of monocytes/macrophages at 24 hours and 5 days. Our studies demonstrate that COX products may serve as endogenous repressors of MCP-1 formation in experimental glomerulonephritis. The data suggest that COX-1 and COX-2 products mediate these effects differently because the selective COX-2 inhibitors had less influence on chemokine expression.

  9. Monocyte chemoattractant protein-induced protein 1 targets hypoxia-inducible factor 1α to protect against hepatic ischemia/reperfusion injury.

    PubMed

    Sun, Peng; Lu, Yue-Xin; Cheng, Daqing; Zhang, Kuo; Zheng, Jilin; Liu, Yupeng; Wang, Xiaozhan; Yuan, Yu-Feng; Tang, Yi-Da

    2018-05-09

    Sterile inflammation is an essential factor causing hepatic ischemia/reperfusion (I/R) injury. As a critical regulator of inflammation, the role of monocyte chemoattractant protein-induced protein 1 (MCPIP1) in hepatic I/R injury remains undetermined. In this study, we discovered that MCPIP1 downregulation was associated with hepatic I/R injury in liver transplant patients and a mouse model. Hepatocyte-specific Mcpip1 gene knockout (HKO) and transgenic (HTG) mice demonstrated that MCPIP1 functions to ameliorate liver damage, reduce inflammation, prevent cell death, and promote regeneration. A mechanistic study revealed that MCPIP1 interacted with and maintained hypoxia-inducible factor 1α (HIF-1α) expression by deubiquitinating HIF-1α. Notably, HIF-1α inhibitor reversed the protective effect of MCPIP1, while HIF-1α activator compensated for the detrimental effect of MCPIP1 deficiency. Thus, we identified the MCPIP1-HIF-1α axis as a critical pathway that may be a good target for intervention in hepatic I/R injury. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  10. Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morooka, Nobukatsu, E-mail: amorooka@gunma-u.ac.jp; Ueguri, Kei; Yee, Karen Kar Lye

    Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatorymore » macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue. - Highlights: • DHT, non-aromatizable androgen suppresses Mcp-1 expression in adipocytes. • Mcp-1 transcription was negatively regulated by DHT-AR action. • DHT-AR selectively regulates Mcp-1 transcription through distinct NF-κB sites.« less

  11. Curcumin as a natural regulator of monocyte chemoattractant protein-1.

    PubMed

    Karimian, Maryam Saberi; Pirro, Matteo; Majeed, Muhammed; Sahebkar, Amirhossein

    2017-02-01

    Monocyte chemoattractant/chemotactic protein-1 (MCP-1), a member of the CC chemokine family, is one of the key chemokines that regulate migration and tissue infiltration of monocytes/macrophages. Its role in the pathophysiology of several inflammatory diseases has been widely recognized, thus making MCP-1 a possible target for anti-inflammatory treatments. Curcumin (diferuloylmethane) is a natural polyphenol derived from the rhizomes of Curcuma Longa L. (turmeric). Anti-inflammatory action underlies numerous pharmacological effects of curcumin in the control and prevention of several diseases. The purpose of this review is to evaluate the effects of curcumin on the regulation of MCP-1 as a key mediator of chemotaxis and inflammation, and the biological consequences thereof. In vitro studies have shown that curcumin can decrease MCP-1 production in various cell lines. Animal studies have also revealed that curcumin can attenuate MCP-1 expression and improve a range of inflammatory diseases through multiple molecular targets and mechanisms of action. There is limited data from human clinical trials showing the decreasing effect of curcumin on MCP-1 concentrations and improvement of the course of inflammatory diseases. Most of the in vitro and animal studies confirm that curcumin exert its MCP-1-lowering and anti-inflammatory effects by down-regulating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway. As yet, there is limited data from human clinical trials showing the effect of curcumin on MCP-1 levels and improvement of the course of inflammatory diseases. More evidence, especially from human studies, is needed to better assess the effects of curcumin on circulating MCP-1 in different human diseases and the role of this modulatory effect in the putative anti-inflammatory properties of curcumin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Phyllostachys edulis Compounds Inhibit Palmitic Acid-Induced Monocyte Chemoattractant Protein 1 (MCP-1) Production

    PubMed Central

    Higa, Jason K.; Liang, Zhibin; Williams, Philip G.; Panee, Jun

    2012-01-01

    Background Phyllostachys edulis Carriere (Poaceae) is a bamboo species that is part of the traditional Chinese medicine pharmacopoeia. Compounds and extracts from this species have shown potential applications towards several diseases. One of many complications found in obesity and diabetes is the link between elevated circulatory free fatty acids (FFAs) and chronic inflammation. This study aims to present a possible application of P. edulis extract in relieving inflammation caused by FFAs. Monocyte chemoattractant protein 1 (MCP-1/CCL2) is a pro-inflammatory cytokine implicated in chronic inflammation. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) are transcription factors activated in response to inflammatory stimuli, and upregulate pro-inflammatory cytokines such as MCP-1. This study examines the effect of P. edulis extract on cellular production of MCP-1 and on the NF-κB and AP-1 pathways in response to treatment with palmitic acid (PA), a FFA. Methodology/Principal Findings MCP-1 protein was measured by cytometric bead assay. NF-κB and AP-1 nuclear localization was detected by colorimetric DNA-binding ELISA. Relative MCP-1 mRNA was measured by real-time quantitative PCR. Murine cells were treated with PA to induce inflammation. PA increased expression of MCP-1 mRNA and protein, and increased nuclear localization of NF-κB and AP-1. Adding bamboo extract (BEX) inhibited the effects of PA, reduced MCP-1 production, and inhibited nuclear translocation of NF-κB and AP-1 subunits. Compounds isolated from BEX inhibited MCP-1 secretion with different potencies. Conclusions/Significance PA induced MCP-1 production in murine adipose, muscle, and liver cells. BEX ameliorated PA-induced production of MCP-1 by inhibiting nuclear translocation of NF-κB and AP-1. Two O-methylated flavones were isolated from BEX with functional effects on MCP-1 production. These results may represent a possible therapeutic

  13. Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbeta1 in melanoma enhances tumor infiltration and immunosuppression.

    PubMed

    Díaz-Valdés, Nancy; Basagoiti, María; Dotor, Javier; Aranda, Fernando; Monreal, Iñaki; Riezu-Boj, José Ignacio; Borrás-Cuesta, Francisco; Sarobe, Pablo; Feijoó, Esperanza

    2011-02-01

    Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFβ1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFβ1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFβ1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFβ1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,β1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFβ1-blocking peptide P144, TGFβ1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFβ1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma.

  14. Monocyte recruitment and expression of monocyte chemoattractant protein-1 are developmentally regulated in remodeling bone in the mouse.

    PubMed Central

    Volejnikova, S.; Laskari, M.; Marks, S. C.; Graves, D. T.

    1997-01-01

    Tooth eruption is defined as the movement of a tooth from its site of development within the alveolar bone to its position of function in the oral cavity. It represents an excellent model to examine osseous metabolism as bone resorption and bone formation occur simultaneously and are spatially separated. Bone resorption occurs in the coronal (occlusal) area, whereas bone formation occurs in the basal area. Monocytes are thought to have a significant role in the regulation of osseous metabolism. The goal of this study was to examine the recruitment of monocytes to bone in C57BL/6J mice that are undergoing developmentally regulated bone remodeling. Monocytes were detected by immunohistochemistry and osteoclasts were counted as bone-associated multi-nucleated, tartrate-resistant acid phosphatase (TRAP)-positive cells. Cell numbers were obtained from histological sections of animals sacrificed daily for 14 days after birth; an image analysis system was used for quantification. The results demonstrated that, immediately after birth, there were relatively few monocytic cells. In the area of bone resorption, the number of monocytes increased with time, reaching peaks at 5 and 9 days, and decreased thereafter. A similar pattern was observed for osteoclasts. In the area of bone formation, there was a time-dependent increase in the number of monocytes. In contrast, the number of osteoclasts in this area was highest at the earliest time points and decreased after day 3. To investigate potential mechanisms for the recruitment of monocytes, expression of monocyte chemoattractant protein (MCP)-1 was assessed. The number of MCP-1-positive cells increased with time and was generally proportional to the recruitment of mononuclear phagocytes. Osteoblasts were the principal bone cell type expressing MCP-1. The results demonstrate that the recruitment of mononuclear cells in the occlusal area is associated with bone resorption. In contrast, recruitment of monocytes in the basal area

  15. Evaluation of TGF-β1 and MCP-1 expression and tubulointerstitial fibrosis in children with Henoch-Schönlein purpura nephritis and IgA nephropathy: A clinical correlation.

    PubMed

    Shuiai, Zhao; Huijun, Shen; Weizhong, Gu; Aimin, Liu; Jianhua, Mao

    2017-02-01

    Henoch-Schönlein purpura nephritis and immunoglobulin A nephropathy are two diseases with similar clinical presentations but very different prognoses. Transforming growth factor β1 and monocyte chemoattractant protein-1 have been associated with the development of tissue fibrosis. We examined the development of tubulointerstitial fibrosis and its relationship with Transforming growth factor β1 and monocyte chemoattractant protein-1 expression in these patients. Renal tissue samples were collected by renal biopsy from 50 children with Henoch-Schönlein purpura nephritis and 50 children with immunoglobulin A nephropathy. Hematoxylin and eosin and Masson's trichrome-stained tissues were examined using light microscopy. Tubulointerstitial fibrosis was graded using the method described by Bohle et al. (1). The immunohistochemical detection of Transforming growth factor β1 and monocyte chemoattractant protein-1 expression was correlated with the tubulointerstitial fibrosis grade. Clinical Trial registration number: ZJCH-2012-0105. Transforming growth factor β1 and monocyte chemoattractant protein-1 expression in the renal tissues was significantly greater in the patients with immunoglobulin A nephropathy than in the patients with Henoch-Schönlein purpura nephritis (both p<0.001). The immunoglobulin A nephropathy patients had a higher tubulointerstitial fibrosis grade than the Henoch-Schönlein purpura nephritis patients (p<0.001). The tubulointerstitial fibrosis grade was in accordance with the Transforming growth factor β1 and monocyte chemoattractant protein-1 expression levels in both diseases (both p<0.001). Transforming growth factor β1 and monocyte chemoattractant protein-1 expression was associated with the development of immunoglobulin A nephropathy and Henoch-Schönlein purpura nephritis. Further studies are needed to better evaluate this association.

  16. The Monocyte Chemoattractant Protein-1/Cognate CC Chemokine Receptor 2 System Affects Cell Motility in Cultured Human Podocytes

    PubMed Central

    Burt, Davina; Salvidio, Gennaro; Tarabra, Elena; Barutta, Federica; Pinach, Silvia; Dentelli, Patrizia; Camussi, Giovanni; Perin, Paolo Cavallo; Gruden, Gabriella

    2007-01-01

    In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases. PMID:18055544

  17. Loss of PPAR gamma in immune cells impairs the ability of abscisic acid to improve insulin sensitivity by suppressing monocyte chemoattractant protein-1 expression and macrophage infiltration into white adipose tissue.

    PubMed

    Guri, Amir J; Hontecillas, Raquel; Ferrer, Gerardo; Casagran, Oriol; Wankhade, Umesh; Noble, Alexis M; Eizirik, Decio L; Ortis, Fernanda; Cnop, Miriam; Liu, Dongmin; Si, Hongwei; Bassaganya-Riera, Josep

    2008-04-01

    Abscisic acid (ABA) is a natural phytohormone and peroxisome proliferator-activated receptor gamma (PPARgamma) agonist that significantly improves insulin sensitivity in db/db mice. Although it has become clear that obesity is associated with macrophage infiltration into white adipose tissue (WAT), the phenotype of adipose tissue macrophages (ATMs) and the mechanisms by which insulin-sensitizing compounds modulate their infiltration remain unknown. We used a loss-of-function approach to investigate whether ABA ameliorates insulin resistance through a mechanism dependent on immune cell PPARgamma. We characterized two phenotypically distinct ATM subsets in db/db mice based on their surface expression of F4/80. F4/80(hi) ATMs were more abundant and expressed greater concentrations of chemokine receptor (CCR) 2 and CCR5 when compared to F4/80(lo) ATMs. ABA significantly decreased CCR2(+) F4/80(hi) infiltration into WAT and suppressed monocyte chemoattractant protein-1 (MCP-1) expression in WAT and plasma. Furthermore, the deficiency of PPARgamma in immune cells, including macrophages, impaired the ability of ABA to suppress the infiltration of F4/80(hi) ATMs into WAT, to repress WAT MCP-1 expression and to improve glucose tolerance. We provide molecular evidence in vivo demonstrating that ABA improves insulin sensitivity and obesity-related inflammation by inhibiting MCP-1 expression and F4/80(hi) ATM infiltration through a PPARgamma-dependent mechanism.

  18. Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells

    PubMed Central

    Hao, Wen-Rui; Sung, Li-Chin; Chen, Chun-Chao; Chen, Jin-Jer

    2018-01-01

    Moderate coffee consumption is inversely associated with cardiovascular disease mortality; however, mechanisms underlying this causal effect remain unclear. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. This study investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion in vascular endothelial cells. Cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinases (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase and p38 MAPK. By contrast, cafestol upregulated cyclic-strain-induced HO-1 and Sirt1 expression. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly attenuated cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. This is the first study to report that cafestol inhibited cyclic-strain-induced inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol. PMID:29854096

  19. Monocyte Chemoattractant Protein-1 in the choroid plexus: a potential link between vascular pro-inflammatory mediators and the CNS during peripheral tissue inflammation

    PubMed Central

    Mitchell, K.; Yang, H.-Y. T.; Berk, J. D.; Tran, J. H.; Iadarola, M. J.

    2009-01-01

    During peripheral tissue inflammation, inflammatory processes in the CNS can be initiated by blood-borne pro-inflammatory mediators. The choroid plexus, the site of CSF production, is a highly specialized interface between the vascular system and CNS, and thus, this structure may be an important element in communication between the vascular compartment and the CNS during peripheral tissue inflammation. We investigated the potential participation of the choroid plexus in this process during peripheral tissue inflammation by examining expression of the SCYA2 gene which codes for monocyte chemoattractant protein-1 (MCP-1). MCP-1 protein was previously reported to be induced in a variety of cells during peripheral tissue inflammation. In the basal state, SCYA2 is highly expressed in the choroid plexus as compared to other CNS tissues. During hind paw inflammation, SCYA2 expression was significantly elevated in choroid plexus, whereas it remained unchanged in a variety of brain regions. The SCYA2-expressing cells were strongly associated with the choroid plexus as vascular depletion of blood cells by whole-body saline flush did not significantly alter SCYA2 expression in the choroid plexus. In situ hybridization suggested that the SCYA2-expressing cells were localized to the choroid plexus stroma. To elucidate potential molecular mechanisms of SCYA2 increase, we examined genes in the NF-κβ signaling cascade including TNF-α, IL-1β and IκBα in choroid tissue. Given that we also detected increased levels of MCP-1 protein by ELISA, we sought to identify potential downstream targets of MCP-1 and observed altered expression levels of mRNAs encoding tight junction proteins TJP2 and claudin 5. Finally, we detected a substantial up-regulation of the transcript encoding E-selectin, a molecule which could participate in leukocyte recruitment to the choroid plexus along with MCP-1. Together, these results suggest that profound changes occur in the choroid plexus during

  20. The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin permeability.

    PubMed

    Lee, Eun Young; Chung, Choon Hee; Khoury, Charbel C; Yeo, Tet Kin; Pyagay, Petr E; Wang, Amy; Chen, Sheldon

    2009-07-01

    The role of monocyte chemoattractant protein-1 (MCP-1) in diabetic nephropathy is typically viewed through the lens of inflammation, but MCP-1 might exert noninflammatory effects on the kidney cells directly. Glomerular podocytes in culture, verified to express the marker nephrin, were exposed to diabetic mediators such as high glucose or angiotensin II and assayed for MCP-1. Only transforming growth factor-beta (TGF-beta) significantly increased MCP-1 production, which was prevented by SB431542 and LY294002, indicating that signaling proceeded through the TGF-beta type I receptor kinase and the phosphatidylinositol 3-kinase pathway. The TGF-beta-induced MCP-1 was found to activate the podocyte's cysteine-cysteine chemokine receptor 2 (CCR2) and, as a result, enhance the cellular motility, cause rearrangement of the actin cytoskeleton, and increase podocyte permeability to albumin in a Transwell assay. The preceding effects of TGF-beta were replicated by treatment with recombinant MCP-1 and blocked by a neutralizing anti-MCP-1 antibody or a specific CCR2 inhibitor, RS102895. In conclusion, this is the first description that TGF-beta signaling through PI3K induces the podocyte expression of MCP-1 that can then operate via CCR2 to increase cellular migration and alter albumin permeability characteristics. The pleiotropic effects of MCP-1 on the resident kidney cells such as the podocyte may exacerbate the disease process of diabetic albuminuria.

  1. Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

    PubMed

    Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

    2015-01-01

    Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

  2. Ascorbic acid deficiency stimulates hepatic expression of inflammatory chemokine, cytokine-induced neutrophil chemoattractant-1, in scurvy-prone ODS rats.

    PubMed

    Horio, Fumihiko; Kiyama, Keiichiro; Kobayashi, Misato; Kawai, Kaori; Tsuda, Takanori

    2006-02-01

    ODS rat has a hereditary defect in ascorbic acid biosynthesis and is a useful animal model for elucidating the physiological role of ascorbic acid. We previously demonstrated by using ODS rats that ascorbic acid deficiency changes the hepatic gene expression of acute phase proteins, as seen in acute inflammation. In this study, we investigated the effects of ascorbic acid deficiency on the production of inflammatory chemokine, cytokine-induced neutrophil chemoattractant-1 (CINC-1), in ODS rats. Male ODS rats (6 wk of age) were fed a basal diet containing ascorbic acid (300 mg/kg diet) or a diet without ascorbic acid for 14 d. Obvious symptoms of scurvy were not observed in the ascorbic acid-deficient rats. Ascorbic acid deficiency significantly elevated the serum concentration of CINC-1 on d 14. The liver and spleen CINC-1 concentrations in the ascorbic acid-deficient rats were significantly elevated to 600% and 180% of the respective values in the control rats. However, the lung concentration of CINC-1 was not affected by ascorbic acid deficiency. Ascorbic acid deficiency significantly elevated the hepatic mRNA level of CINC-1 (to 480% of the value in the control rats), but not the lung mRNA level. These results demonstrate that ascorbic acid deficiency elevates the serum, liver and spleen concentrations of CINC-1 as seen in acute inflammation, and suggest that ascorbic acid deficiency stimulate the hepatic CINC-1 gene expression.

  3. Plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, e-selectin and C-reactive protein levels in response to 4-week very-high-fructose or -glucose diets.

    PubMed

    Silbernagel, G; Machann, J; Häring, H-U; Fritsche, A; Peter, A

    2014-01-01

    High intake of added sweeteners is considered to have a causal role in the pathogenesis of cardiometabolic disorders. Especially, high-fructose intake is regarded as potentially harmful to cardiometabolic health. It may cause not only weight gain but also low-grade inflammation, which represents an independent risk factor for developing type 2 diabetes and cardiovascular disease. In particular, fructose has been suggested to induce plasminogen activator inhibitor-1 (PAI-1) expression in the liver and to increase circulating inflammatory cytokines. We therefore aimed to investigate, whether high-fructose diet has an impact on PAI-1, monocyte chemoattractant protein-1 (MCP-1), e-selectin and C-reactive protein (CRP) concentrations in healthy humans. We studied 20 participants (12 males and 8 females) of the TUebingen FRuctose Or Glucose study. This is an exploratory, parallel, prospective, randomized, single-blinded, outpatient, hypercaloric, intervention study. The participants had a mean age of 30.9 ± 2.1 years and a mean body mass index of 26.0 ± 0.5 kg/m(2) and they received 150 g of either fructose or glucose per day for 4 weeks. There were neither significant changes of PAI-1, MCP-1, e-selectin and CRP after fructose (n=10) and glucose (n=10) intervention nor treatment effects (all P>0.2). Moreover, we did not observe longitudinal associations of the inflammatory parameters with triglycerides, liver fat, visceral fat and body weight in the fructose group. Temporary high-fructose intake does not seem to cause inflammation in apparently healthy people in this secondary analysis of a small feeding trial.

  4. Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

    PubMed

    Orellana, Renan; Kato, Sumie; Erices, Rafaela; Bravo, María Loreto; Gonzalez, Pamela; Oliva, Bárbara; Cubillos, Sofía; Valdivia, Andrés; Ibañez, Carolina; Brañes, Jorge; Barriga, María Isabel; Bravo, Erasmo; Alonso, Catalina; Bustamente, Eva; Castellon, Enrique; Hidalgo, Patricia; Trigo, Cesar; Panes, Olga; Pereira, Jaime; Mezzano, Diego; Cuello, Mauricio A; Owen, Gareth I

    2015-04-15

    An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

  5. A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

    PubMed Central

    Chen, Mei-Yu; Long, Yu; Devreotes, Peter N.

    1997-01-01

    Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA− null mutants, neither chemoattractant stimulation of intact cells nor GTPγS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPγS stimulation of adenylyl cyclase activity in piaA− lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac− piaA− double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. PMID:9389653

  6. MCP-1: chemoattractant with a role beyond immunity: a review.

    PubMed

    Yadav, Amita; Saini, Vandana; Arora, Sarika

    2010-11-11

    Monocyte Chemoattractant Protein (MCP)-1, a potent monocyte attractant, is a member of the CC chemokine subfamily. MCP-1 exerts its effects through binding to G-protein-coupled receptors on the surface of leukocytes targeted for activation and migration. Role of MCP-1 and its receptor CCR2 in monocyte recruitment during infection or under other inflammatory conditions is well known. A comprehensive literature search was conducted from the websites of the National Library of Medicine (http://www.ncbl.nlm.nih.gov) and Pubmed Central, the US National Library of Medicine's digital archive of life sciences literature (http://www.pubmedcentral.nih.gov/). The data was assessed from books and journals that published relevant articles in this field. Recent and ongoing research indicates the role of MCP-1 in various allergic conditions, immunodeficiency diseases, bone remodelling, and permeability of blood - brain barrier, atherosclerosis, nephropathies and tumors. MCP-1 plays an important role in pathogenesis of various disease states and hence MCP-1 inhibition may have beneficial effects in such conditions. 2010 Elsevier B.V. All rights reserved.

  7. Effects of High Estrogen Levels on Monocyte Chemoattractant Protein-1 and Wound Healing

    PubMed Central

    Plackett, Timothy P.; Gregory, Meredith S.; Kovacs, Elizabeth J.

    2015-01-01

    Objective: Herein, we tested the effects of high levels of supplemental estrogen treatment on cutaneous wound healing. Approach: Female mice were implanted with a 17β-estradiol (E2) secreting pellet or placebo before receiving a full-thickness dermal excisional wound. Mice receiving the E2 pellet attained hormone levels that are comparable to those achieved during pregnancy. At 1, 3, and 5 days after injury, the dermal excision wound was examined for their histologic appearance, rate of closure, and chemokine levels. Results: Wound closure, assessed by percent reepithelialization, was slower in E2-treated mice relative to placebo (42.6%±6.6% vs. 70.0%±5.3%, respectively, 3 days after injury). In addition, there was a marked reduction in the subepithelial inflammatory infiltrate and granulation tissue in E2-treated mice relative to placebo. Wound levels of monocyte chemoattractant protein-1 (MCP-1) were increased by 3 days after injury and continued to rise at 5 days after injury in placebo-treated mice (p<0.01). By contrast, MCP-1 levels were significantly reduced at 3 and 5 days after injury in E2-treated mice relative to placebo-treated controls (p<0.01). This attenuation could be reversed by treatment with an estrogen receptor antagonist. Innovation: High levels of estrogen are able to suppress normal wound closure. Conclusion: Dermal wound healing can be altered by manipulating the gonadal steroid hormone levels. In particular, high levels of estrogen can be utilized to slow down the rate of wound healing through a reduction in the inflammatory response. PMID:25713751

  8. An ion mobility-mass spectrometry investigation of monocyte chemoattractant protein-1

    NASA Astrophysics Data System (ADS)

    Schenauer, Matthew R.; Leary, Julie A.

    2009-10-01

    In the present article we describe the gas-phase dissociation behavior of the dimeric form of monocyte chemoattractant protein-1 (MCP-1) using quadrupole-traveling wave ion mobility spectrometry-time of flight mass spectrometry (q-TWIMS-TOF MS) (Waters Synapt(TM)). Through investigation of the 9+ charge state of the dimer, we were able to monitor dissociation product ion (monomer) formation as a function of activation energy. Using ion mobility, we were able to observe precursor ion structural changes occurring throughout the activation process. Arrival time distributions (ATDs) for the 5+ monomeric MCP-1 product ions, derived from the gas-phase dissociation of the 9+ dimer, were then compared with ATDs obtained for the 5+ MCP-1 monomer isolated directly from solution. The results show that the dissociated monomer is as compact as the monomer arising from solution, regardless of the trap collision energy (CE) used in the dissociation. The solution-derived monomer, when collisionally activated, also resists significant unfolding within measure. Finally, we compared the collisional activation data for the MCP-1 dimer with an MCP-1 dimer non-covalently bound to a single molecule of the semi-synthetic glycosaminoglycan (GAG) analog Arixtra(TM); the latter a therapeutic anti-thrombin III-activating pentasaccharide. We observed that while dimeric MCP-1 dissociated at relatively low trap CEs, the Arixtra-bound dimer required much higher energies, which also induced covalent bond cleavage in the bound Arixtra molecule. Both the free and Arixtra-bound dimers became less compact and exhibited longer arrival times with increasing trap CEs, albeit the Arixtra-bound complex at slightly higher energies. That both dimers shifted to longer arrival times with increasing activation energy, while the dissociated MCP-1 monomers remained compact, suggests that the longer arrival times of the Arixtra-free and Arixtra-bound dimers may represent a partial breach of non

  9. Mitomycin C upregulates IL-8 and MCP-1 chemokine expression via mitogen-activated protein kinases in corneal fibroblasts.

    PubMed

    Chou, San-Fang; Chang, Shu-Wen; Chuang, Jia-Ling

    2007-05-01

    To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.

  10. Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in patients with amyotrophic lateral sclerosis.

    PubMed

    Martínez, H R; Escamilla-Ocañas, C E; Camara-Lemarroy, C R; González-Garza, M T; Moreno-Cuevas, J; García Sarreón, M A

    2017-10-10

    Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1β levels and assessed their association with the duration and severity of ALS. Concentrations of MCP-1 and MIP-1β were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (<12months vs. >12months) and severity (<30points vs. >30points on the ALS Functional Rating Scale administered at hospital admission). Higher CSF MIP-1β (10.68pg/mL vs. 4.69pg/mL, P<.0001) and MCP-1 (234.89pg/mL vs. 160.95pg/mL, P=.011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1β and MCP-1 in patients with ALS. The increase in MIP-1β and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  11. Cerebrospinal fluid monocyte chemoattractant protein-1 in alcoholics: support for a neuroinflammatory model of chronic alcoholism.

    PubMed

    Umhau, John C; Schwandt, Melanie; Solomon, Matthew G; Yuan, Peixiong; Nugent, Allison; Zarate, Carlos A; Drevets, Wayne C; Hall, Samuel D; George, David T; Heilig, Markus

    2014-05-01

    Liver inflammation in alcoholism has been hypothesized to influence the development of a neuroinflammatory process in the brain characterized by neurodegeneration and altered cognitive function. Monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) elevations have been noted in the alcoholic brain at autopsy and may have a role in this process. We studied cerebrospinal fluid (CSF) levels of MCP-1 as well as interleukin-1β and tumor necrosis factor-α in 13 healthy volunteers and 28 alcoholics during weeks 1 and 4 following detoxification. Serum liver enzymes were obtained as markers of alcohol-related liver inflammation. Compared to healthy volunteers, MCP-1 levels were significantly higher in alcoholics both on day 4 and day 25 (p < 0.0001). Using multiple regression analysis, we found that MCP-1 concentrations were positively associated with the liver enzymes gamma glutamyltransferase (GGT; p = 0.03) and aspartate aminotransferase/glutamic oxaloacetic transaminase (AST/GOT; p = 0.004). These preliminary findings are consistent with the hypothesis that neuroinflammation as indexed by CSF MCP-1 is associated with alcohol-induced liver inflammation, as defined by peripheral concentrations of GGT and AST/GOT. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  12. Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

    PubMed

    Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

    2018-07-04

    The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

  13. Mutant monocyte chemoattractant protein 1 protein attenuates migration of and inflammatory cytokine release by macrophages exposed to orthopedic implant wear particles.

    PubMed

    Yao, Zhenyu; Keeney, Michael; Lin, Tzu-Hua; Pajarinen, Jukka; Barcay, Katherine; Waters, Heather; Egashira, Kensuke; Yang, Fan; Goodman, Stuart

    2014-09-01

    Wear particles generated from total joint replacements can stimulate macrophages to release chemokines, such as monocyte chemoattractant protein 1 (MCP-1), which is the most important chemokine regulating systemic and local cell trafficking and infiltration of monocyte/macrophages in chronic inflammation. One possible strategy to curtail the adverse events associated with wear particles is to mitigate migration and activation of monocyte/macrophages. The purpose of this study is to modulate the adverse effects of particulate biomaterials and inflammatory stimuli such as endotoxin by interfering with the biological effects of the chemokine MCP-1. In the current study, the function of MCP-1 was inhibited by the mutant MCP-1 protein called 7ND, which blocks its receptor, the C-C chemokine receptor type 2 (CCR2) on macrophages. Addition of 7ND decreased MCP-1-induced migration of THP-1 cells in cell migration experiments in a dose-dependent manner. Conditioned media from murine macrophages exposed to clinically relevant polymethylmethacrylate (PMMA) particles with/without endotoxin [lipopolysaccharide (LPS)] had a chemotactic effect on human macrophages, which was decreased dramatically by 7ND. 7ND demonstrated no adverse effects on the viability of macrophages, and the capability of mesenchymal stem cells (MSCs) to form bone at the doses tested. Finally, proinflammatory cytokine production was mitigated when macrophages were exposed to PMMA particles with/without LPS in the presence of 7ND. Our studies confirm that the MCP-1 mutant protein 7ND can decrease macrophage migration and inflammatory cytokine release without adverse effects at the doses tested. Local delivery of 7ND at the implant site may provide a therapeutic strategy to diminish particle-associated periprosthetic inflammation and osteolysis. © 2013 Wiley Periodicals, Inc.

  14. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  15. Monocyte Chemoattractant Protein-1 as a Predictor of Coronary Atherosclerosis in Patients Receiving Coronary Angiography.

    PubMed

    Mahler, Simon A; Register, Thomas C; Riley, Robert F; D'Agostino, Ralph B; Stopyra, Jason P; Miller, Chadwick D

    2018-06-01

    Animal studies suggest that monocyte chemoattractant protein-1 (MCP-1) is a promising biomarker for coronary artery atherosclerosis (CAA), but human studies have been inconclusive. To determine potential relationships between plasma MCP-1 and CAA in patients with acute chest pain. A secondary analysis of 150 patients enrolled in emergency department chest pain risk stratification clinical investigations was conducted. Participants with stored blood and known coronary phenotypes (determined by coronary angiography) were selected using stratified randomization such that 50 patients were included into 3 groups: (1) no angiographic evidence of CAA, (2) nonobstructive CAA, and (3) obstructive CAA (stenosis ≥ 70%). Plasma MCP-1 levels were determined by enzyme-linked immunosorbent assay. The association between MCP-1 and obstructive CAA or any CAA was modeled using logistic regression. Variables in the unreduced model included age, sex, race, prior diagnosis of CAA or acute coronary syndrome, hyperlipidemia, hypertension, diabetes, smoking, and cardiac troponin I measurement. Among the 150 participants, 65.3% (98/150) had invasive coronary angiography and 34.7% (52/150) had coronary computed tomographic angiography. Myocardial infarction occurred in 27.3% (41/150) and coronary revascularization occurred in 26% (39/150) of the participants. Each 10 pg/mL increase in MCP-1 measurement was associated with an odds ratio of 1.12 (95% confidence interval, 1.06-1.19) for obstructive CAA. MCP-1 remained a significant predictor of obstructive CAA and any CAA after adjustment for age, sex, race, traditional cardiac risk factors, and cardiac troponin I. MCP-1 is independently associated with CAA among emergency department patients with chest pain.

  16. Association of Monocyte Chemoattractant Protein-1 with Death and Atherosclerotic Events in Chronic Kidney Disease.

    PubMed

    Gregg, L Parker; Tio, Maria Clarissa; Li, Xilong; Adams-Huet, Beverley; de Lemos, James A; Hedayati, S Susan

    2018-06-06

    Monocyte chemoattractant protein-1 -(MCP-1), a marker of inflammation and monocyte recruitment to atherosclerotic plaques, is associated with cardiovascular (CV) outcomes in patients with acute coronary syndrome. Although plasma levels are elevated in chronic kidney disease (CKD), associations with reduced kidney function or outcomes in CKD have not been explored. In this population-based, probability-sampled, longitudinal cohort of 3,257 participants, including 286 (8.8%) patients with CKD, we studied the association of plasma MCP-1 with estimated glomerular filtration rate (eGFR), albuminuria, death, and intermediate and hard CV outcomes in CKD and non-CKD individuals. Cox proportional hazards regression assessed associations of baseline MCP-1 with all-cause death and atherosclerotic events. MCP-1 was higher in CKD than non-CKD participants (p < 0.001), and negatively associated with eGFR (r = -0.23, p < 0.0001) but not albuminuria in CKD. MCP-1 was associated with pulse wave velocity and coronary artery calcification in non-CKD but not CKD individuals. At 13.5 years, there were 230 (7.7%) deaths and 168 (6.4%) atherosclerotic events in the non-CKD vs. 97 (34.0%) deaths and 62 (27.9%) events in the CKD group (p < 0.001 for each). MCP-1 was associated with death (hazards ratio [HR] 2.0 [1.4-2.9] per log-unit increase) and atherosclerotic events (1.7 [1.0-2.9]) in CKD individuals. The HR for death in CKD remained significant (1.6 [1.1-2.3]) after adjusting for CV risk factors. Although plasma MCP-1 increased with decreased eGFR, it remained an independent risk factor for death in CKD. MCP-1 did not correlate with intermediate CV outcomes, implicating pathways other than atherosclerosis in the association of MCP-1 with death in CKD. © 2018 S. Karger AG, Basel.

  17. Identification of serum monocyte chemoattractant protein-1 and prolactin as potential tumor markers in hepatocellular carcinoma.

    PubMed

    Wang, Who-Whong; Ang, Soo Fan; Kumar, Rajneesh; Heah, Charmain; Utama, Andi; Tania, Navessa Padma; Li, Huihua; Tan, Sze Huey; Poo, Desmond; Choo, Su Pin; Chow, Wan Cheng; Tan, Chee Kiat; Toh, Han Chong

    2013-01-01

    Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p<0.001). In conclusion, prolactin and MCP-1 are overexpressed in HCC and are conveniently quantifiable in patients' sera by ELISA. MCP-1 appears to be a promising complementary biomarker for HCC diagnosis and this MCP-1+AFP model should be further evaluated as potential

  18. Identification of Serum Monocyte Chemoattractant Protein-1 and Prolactin as Potential Tumor Markers in Hepatocellular Carcinoma

    PubMed Central

    Kumar, Rajneesh; Heah, Charmain; Utama, Andi; Tania, Navessa Padma; Li, Huihua; Tan, Sze Huey; Poo, Desmond; Choo, Su Pin; Chow, Wan Cheng; Tan, Chee Kiat; Toh, Han Chong

    2013-01-01

    Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p<0.001). In conclusion, prolactin and MCP-1 are overexpressed in HCC and are conveniently quantifiable in patients’ sera by ELISA. MCP-1 appears to be a promising complementary biomarker for HCC diagnosis and this MCP-1+AFP model should be further evaluated as potential

  19. Lipopolysaccharide and hypoxia significantly alters interleukin-8 and macrophage chemoattractant protein-1 production by human fibroblasts but not fibrosis related factors.

    PubMed

    Eleftheriadis, T; Liakopoulos, V; Lawson, B; Antoniadi, G; Stefanidis, I; Galaktidou, G

    2011-07-01

    Besides extracellular matrix production, fibroblasts are able to produce various cytokines. Their ubiquitous position makes fibroblasts appropriate cells for sensing various noxious stimuli and for attracting immune cells in the affected area. In the present study the effect of lipopolysaccharide (LPS) and cobalt chloride (CoCl(2)) on the above fibroblasts functions were evaluated in primary human skin fibroblasts cultures. Collagen, matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1, transforming growth factor-β1, interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were measured in fibroblasts culture supernatants. Fibroblasts proliferation and viability were assessed as well. Hypoxia inducible factor-1α and the phosphorylated p65 portion of NF-κB were assessed in fibroblasts protein extracts. LPS and CoCl(2) had a minor effect on fibrosis related factors in human primary fibroblasts, possibly due to the absence of interplay with other cell types in the used experimental system. On the contrary both LPS and CoCl(2) increased significantly IL-8. LPS also increased considerably MCP-1, but CoCl(2) decreased it. Thus LPS and CoCl(2) induce a sentinel, nevertheless not identical, phenotype in primary human fibroblasts. The last disparity could result in different body response to infectious or hypoxic noxious stimuli.

  20. Prokaryotic expression and in vitro functional analysis of IL-1β and MCP-1 from guinea pig.

    PubMed

    Dirisala, Vijaya R; Jeevan, Amminikutty; Ly, Lan H; McMurray, David N

    2013-06-01

    The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

  1. Monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in exudative age-related macular degeneration.

    PubMed

    Jonas, Jost B; Tao, Yong; Neumaier, Michael; Findeisen, Peter

    2010-10-01

    To examine intraocular concentrations of monocyte chemoattractant protein 1 (MCP-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and vascular endothelial growth factor (VEGF) in eyes with exudative age-related macular degeneration (AMD). The investigation included a study group of 28 patients (28 eyes) with exudative AMD and a control group of 25 patients (25 eyes) with cataract. The concentrations of MCP-1, sICAM-1, sVCAM-1, and VEGF in aqueous humor samples obtained during surgery were measured using a solid-phase chemiluminescence immunoassay. The study group as compared with the control group had higher aqueous concentrations of sICAM-1 (mean [SD], 844 [2073] vs 246 [206] pg/mL, respectively; P < .001), sVCAM-1 (mean [SD], 7978 [7120] vs 2999 [1426] pg/mL, respectively; P < .001), and MCP-1 (mean [SD], 587 [338] vs 435 [221] pg/mL, respectively; P = .07). The concentration of VEGF did not vary significantly between the groups (P = .76). The MCP-1 concentration was significantly associated with macular thickness (r = 0.40; P = .004). It decreased significantly with the type of subfoveal neovascular membrane (classic membrane type, occult membrane, retinal pigment epithelium detachment) (P = .009). The concentrations of sICAM-1, sVCAM-1, and VEGF were not significantly associated with membrane type and macular thickness (P ≥ .18). Concentrations of MCP-1, sICAM-1, and sVCAM-1 are significantly associated with exudative AMD, even in the presence of normal VEGF concentrations. Intraocular MCP-1 concentrations are correlated with the subfoveal neovascular membrane type and the amount of macular edema. One may infer that MCP-1, sICAM-1, and sVCAM-1 could potentially be additional target molecules in therapy for exudative AMD.

  2. Sex steroid-induced changes in circulating monocyte chemoattractant protein-1 levels may contribute to metabolic dysfunction in obese men.

    PubMed

    Ruige, Johannes B; Bekaert, Marlies; Lapauw, Bruno; Fiers, Tom; Lehr, Stefan; Hartwig, Sonja; Herzfeld de Wiza, Daniella; Schiller, Martina; Passlack, Waltraud; Van Nieuwenhove, Yves; Pattyn, Piet; Cuvelier, Claude; Taes, Youri E; Sell, Henrike; Eckel, Juergen; Kaufman, Jean-Marc; Ouwens, D Margriet

    2012-07-01

    Low testosterone accompanied by elevated estradiol associates with the development of metabolic dysfunction in men. The aim of the study was to explore the hypothesis that alterations in sex steroid levels induce metabolic dysfunction through adipokines. Circulating levels of sex steroids and 28 adipokines were determined in a cross-sectional study of morbidly obese men and aged-matched controls, as well as in a randomized clinical trial with healthy young men in which obesity-related alterations in sex steroid levels were mimicked by treatment with an aromatase inhibitor plus estradiol patches. Morbidly obese men had lower testosterone levels than normal-weight controls. Estradiol levels were increased in morbidly obese men (without DM2) as compared to normal-weight controls. Circulating levels of multiple proinflammatory cytokines, including IL-1Ra, IL-5, IL-6, IL-10, leptin, monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein 1α, positively associated with estradiol and negatively with testosterone. The associations with estradiol, but not with testosterone, remained significant after adjusting for adipocyte cell size. In a separate clinical trial, the direct adverse effects of lowering testosterone and raising estradiol on MCP1 were substantiated in vivo. Initial alterations in sex steroid levels may contribute to metabolic dysfunction through adverse effects on adipokine levels in obese men. The direct adverse effects on MCP1, a chemokine highly linked to the development of metabolic dysfunction, were substantiated in a trial mimicking obesity-related alterations of sex steroid levels in healthy young males.

  3. Dexamethasone reduces mitomycin C-related inflammatory cytokine expression without inducing further cell death in corneal fibroblasts.

    PubMed

    Chang, Shu-Wen; Chou, San-Fang; Yu, Shuen-Yuen

    2010-01-01

    The purpose of this study was to investigate the effect of dexamethasone (DEX) on mitomycin C (MMC)-induced inflammatory cytokine expression in corneal fibroblasts. Primary human corneal fibroblasts were treated with MMC, dexamethasone, or in combination. Morphological changes and cell growth were documented using phase-contrast microscopy and PicoGreen assay, respectively. Cell apoptosis was evaluated by annexin V/propidium iodide staining, whereas viability was tested by the live/dead assay and analyzed by flow cytometry. The relative expression of interleukin-8 and monocyte chemoattractant protein-1 was investigated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Mitogen-activated protein kinase activation and mitogen-activated protein kinase phosphatase-1 expression were documented by Western blot analysis. We found that MMC induced corneal fibroblast elongation, apoptosis, and retarded cell growth, whereas DEX did not significantly alter cell morphology or viability. The combination of DEX and MMC did not induce additional apoptosis and cell death. DEX dose dependently down-regulated basal and MMC-induced interleukin-8 and monocyte chemoattractant protein-1 mRNA expression and protein secretion. DEX attenuated MMC-induced p38 and Jun N-terminal kinases activation and up-regulated expression. These suggested that DEX may inhibit MMC-induced interleukin-8 and monocyte chemoattractant protein-1 by up-regulating MKP-1 expression, which subsequently deactivated p38 and Jun N-terminal kinases activation. Combined MMC and DEX treatment may facilitate corneal wound healing.

  4. Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

    PubMed

    Lahm, H; Hoeflich, A; Andre, S; Sordat, B; Kaltner, H; Wolf, E; Gabius, H J

    2000-09-01

    The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

  5. Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

    PubMed

    Tyrrell, Christine; De Cecco, Martin; Reynolds, Natalie L; Kilanowski, Fiona; Campopiano, Dominic; Barran, Perdita; Macmillan, Derek; Dorin, Julia R

    2010-03-01

    Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis. Copyright 2009 Elsevier Ltd. All rights reserved.

  6. Periodontal regeneration via chemo-attractive constructs.

    PubMed

    Cai, Xinjie; Yang, Fang; Walboomers, X Frank; Wang, Yining; Jansen, John A; van den Beucken, Jeroen J J P; Plachokova, Adelina S

    2018-05-19

    Chemo-attractants, such as stromal cell-derived factor-1α (SDF-1α), can offer an advantage for periodontal regeneration by recruiting the patient's own stem cells to stimulate self-repair. We here developed a chemo-attractive construct for periodontal regeneration using SDF-1α and evaluated its efficacy in vivo. SDF-1α was loaded on gelatin sponge and tested in vitro for SDF-1α release. Subsequently, SDF-1α constructs were implanted into rat periodontal defects for 1 and 6 weeks, with unloaded materials and empty defects as controls. The regenerative efficacy was evaluated by micro-CT, histological and histomorphometrical analyses. In vitro results showed limited SDF-1α release up to 35 days. In contrast, SDF-1α constructs significantly improved periodontal defect regeneration in terms of alveolar bone height, new bone area, and functional ligament length. Additionally, SDF-1α constructs decreased the inflammatory response at week 6. Chemo-attractive constructs significantly improved periodontal regeneration in terms of alveolar bone height, new bone area, and functional ligament length. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Defensin-related peptide 1 (Defr1) is allelic to Defb8 and chemoattracts immature DC and CD4+ T cells independently of CCR6.

    PubMed

    Taylor, Karen; Rolfe, Mark; Reynolds, Natalie; Kilanowski, Fiona; Pathania, Uday; Clarke, Dave; Yang, De; Oppenheim, Joost; Samuel, Kay; Howie, Sarah; Barran, Perdita; Macmillan, Derek; Campopiano, Dominic; Dorin, Julia

    2009-05-01

    Beta-defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. In other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine beta-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4(+) T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines restored (Defr1 Y5C) or with only a single cysteine (Defr1-1c(V)) chemoattract CD4(+) T cells with reduced activity, but do not chemoattract DC. Beta-defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of beta-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6.

  8. DNA methylation of tumor necrosis factor-α, monocyte chemoattractant protein-1, and adiponectin genes in visceral adipose tissue is related to type 2 diabetes in the Xinjiang Uygur population.

    PubMed

    Zhang, Jun; Wang, Cuizhe; Ha, Xiaodan; Li, Wei; Xu, Peng; Gu, Yajuan; Wang, Tingting; Wang, Yan; Xie, Jianxin

    2017-07-01

    The higher probability of type 2 diabetes mellitus (T2DM) in the Uygur population is due to a greater waist:  hip ratio and visceral fat. This study investigated DNA methylation of tumor necrosis factor-α (TNF), monocyte chemoattractant protein-1 (MCP1), and adiponectin (ADIPOQ) in visceral adipose tissue in T2DM. Visceral adipose tissue was collected from Uygur individuals and divided into normal control (NC; n = 50), obese (Ob; n = 48), and T2DM (n = 26) groups. Expression of TNF, ADIPOQ, and MCP1 mRNA and DNA methylation status were quantified by reverse transcription-polymerase chain reaction and denaturing HPLC. The respective methylation-positive rate for ADIPOQ increased gradually from the NC to Ob to T2DM groups (34.0 %, 47.9 %, and 65.4 %; P < 0.05), decreased gradually for TNF (70.0 %, 47.9 %, and 26.9 %; P < 0.01), and did not differ significantly for MCP1 (0 %, 2.08 %, and 0 %). Compared with the NC group, ADIPOQ mRNA expression was significantly lower in the Ob and T2DM groups (median 0.7162 vs 0.4244 and 0.4093, respectively; P < 0.05), whereas TNF and MCP1 expression was significantly higher (median TNF expression: 0.0250 vs 0.1096 and 0.0734 respectively; median MCP1 expression 0.1588 vs 0.1937 and 0.1983, respectively; P < 0.05 for all). Expression of ADIPOQ and TNF was significantly lower in methylation-negative (median 0.7870 and 0.1988, respectively) than methylation-positive (median 0.2700 and 0.0542, respectively) groups (P < 0.01). Lower ADIPOQ and higher TNF and MCP1 mRNA expression in visceral adipose tissue may be correlated with obesity and T2DM in the Uygur population. Promoter DNA methylation affects expression of ADIPOQ and TNF. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  9. Defensin-related peptide 1 (Defr1) is allelic to Defb8 and chemoattracts immature DC and CD4+ T cells independently of CCR6

    PubMed Central

    Taylor, Karen; Rolfe, Mark; Reynolds, Natalie; Kilanowski, Fiona; Pathania, Uday; Clarke, Dave; Yang, De; Oppenheim, Joost; Samuel, Kay; Howie, Sarah; Barran, Perdita; Macmillan, Derek; Campopiano, Dominic; Dorin, Julia

    2009-01-01

    β-Defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine β-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. In other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine β-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4+ T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines restored (Defr1 Y5C) or with only a single cysteine (Defr1-1cV) chemoattract CD4+ T cells with reduced activity, but do not chemoattract DC. β-Defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of β-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6. PMID:19404978

  10. Urine epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors of complete remission in primary glomerulonephritis.

    PubMed

    Chanrat, Eakkapat; Worawichawong, Supanat; Radinahamed, Piyanuch; Sathirapongsasuti, Nuankanya; Nongnuch, Arkom; Assanatham, Montira; Udomsubpayakul, Umaporn; Kitiyakara, Chagriya

    2018-04-01

    The balance of several cytokines likely influences the resolution of glomerulonephritis. Monocyte chemoattractant protein-1(MCP-1) is a chemokine that promotes renal inflammation whereas epidermal growth factor (EGF) stimulates protective responses. Previously, high urine MCP-1(MCP-1) and low urine EGF (EGF) levels were found to be associated with tubulointerstitial fibrosis, but there is limited information on the value of these mediators as predictors of therapeutic responses or long term outcome in primary glomerulonephritis. To determine the performance of urine EGF, MCP-1 or their ratio at baseline as biomarkers to predict complete remission, and the relationship of these mediators with subsequent renal function 24 months later in primary glomerulonephritis. This is a prospective study of patients with biopsy-proven primary glomerulonephritis. Baseline urine samples were collected at biopsy before therapy. MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assays and expressed as a ratio to urine creatinine (ng/mgCr) or as EGF/MCP-1 ratio (ng/ng). Proteinuria and estimated glomerular filtration rate (eGRF) were monitored after therapy. Complete remission (CR) was defined as proteinuria ≤ 0.3 g/gCr. Median follow-up was 20 months. Of all patients (n = 74), 38 patients (51.4%) subsequently achieved CR. Baseline urine EGF and EGF/MCP-1 levels were significantly higher in CR compared to Not CR. By contrast, MCP-1 was not different. High EGF (EGF > 75 ng/mgCr) was a significant predictor (OR 2.28) for CR by multivariate analysis after adjusting for proteinuria, blood pressure, baseline eGFR. In patients who completed 24 months follow-up (n = 43), baseline EGF correlated inversely with proteinuria and positively with eGFR at 24 months. High urine EGF level is a promising biomarker of CR. Baseline EGF levels correlated with kidney function at 2 years. EGF/MCP-1 was not superior to EGF alone. Further studies are necessary to

  11. Hydrogen Sulfide Suppresses Oxidized Low-density Lipoprotein (Ox-LDL)-stimulated Monocyte Chemoattractant Protein 1 generation from Macrophages via the Nuclear Factor κB (NF-κB) Pathway*

    PubMed Central

    Du, Junbao; Huang, Yaqian; Yan, Hui; Zhang, Qiaoli; Zhao, Manman; Zhu, Mingzhu; Liu, Jia; Chen, Stella X.; Bu, Dingfang; Tang, Chaoshu; Jin, Hongfang

    2014-01-01

    This study was designed to examine the role of hydrogen sulfide (H2S) in the generation of oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein 1 (MCP-1) from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS) and hexyl acrylate and then treated with ox-LDL. The results showed that ox-LDL treatment down-regulated the H2S/cystathionine-β-synthase pathway, with increased MCP-1 protein and mRNA expression in both THP-1 cells and RAW macrophages. Hexyl acrylate promoted ox-LDL-induced inflammation, whereas the H2S donor NaHS inhibited it. NaHS markedly suppressed NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter in ox-LDL-treated macrophages. Furthermore, NaHS decreased the ratio of free thiol groups in p65, whereas the thiol reductant DTT reversed the inhibiting effect of H2S on the p65 DNA binding activity. Most importantly, site-specific mutation of cysteine 38 to serine in p65 abolished the effect of H2S on the sulfhydration of NF-κB and ox-LDL-induced NF-κB activation. These results suggested that endogenous H2S inhibited ox-LDL-induced macrophage inflammation by suppressing NF-κB p65 phosphorylation, nuclear translocation, DNA binding activity, and recruitment to the MCP-1 promoter. The sulfhydration of free thiol group on cysteine 38 in p65 served as a molecular mechanism by which H2S inhibited NF-κB pathway activation in ox-LDL-induced macrophage inflammation. PMID:24550391

  12. Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy.

    PubMed

    Tam, Frederick W K; Riser, Bruce L; Meeran, Karim; Rambow, JoAnn; Pusey, Charles D; Frankel, Andrew H

    2009-07-01

    Profibrotic growth factors and inflammatory chemokines have been implicated in the pathogenesis of diabetic nephropathy (DN). However, measurement of urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers has not previously been reported, and neither have two such molecules in urine been examined in a single study of DN. In this prospective observational study, 43 adult diabetic patients were studied, 40 were followed up for 6years. Urinary MCP-1/creatinine ratios were found to be significantly higher in patients with macroalbuminuria (3.3- and 2.1-fold higher (p<0.01) than normoalbuminuric and microalbuminuric patients, respectively). CCN2 exhibited a pattern different from that of urinary MCP-1. Urinary CCN2/creatinine ratios were greatly elevated in both microalbuminuric and macroalbuminuric patients (125- and 74-fold higher than normoalbuminuric patients, respectively, p<0.01 and p<0.05, respectively). Further, urinary CCN2, but not MCP-1, correlated with progression of microalbuminuria (R=0.49, p<0.05). In contrast, MCP-1, but not CCN2, correlated with the rate of eGFR decline for all patients (R=0.61, p<0.0001), reflective of its predictive value in patients with macroalbuminuria, but not for patients with microalbuminuria or normoalbuminuria. In conclusion, increased urinary CCN2 is associated with the early progression of DN, whereas MCP-1 is associated with later stage disease.

  13. The bromodomain protein inhibitor I-BET151 suppresses expression of inflammatory genes and matrix degrading enzymes in rheumatoid arthritis synovial fibroblasts.

    PubMed

    Klein, Kerstin; Kabala, Pawel A; Grabiec, Aleksander M; Gay, Renate E; Kolling, Christoph; Lin, Lih-Ling; Gay, Steffen; Tak, Paul P; Prinjha, Rab K; Ospelt, Caroline; Reedquist, Kris A

    2016-02-01

    To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF). The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays. BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium. Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

    PubMed Central

    Pickford, Fiona; Marcus, Jacob; Camargo, Luiz Miguel; Xiao, Qiurong; Graham, Danielle; Mo, Jan-Rung; Burkhardt, Matthew; Kulkarni, Vinayak; Crispino, Jamie; Hering, Heike; Hutton, Michael

    2011-01-01

    Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β. PMID:21224065

  15. Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1α and MCP-1

    PubMed Central

    Carollo, Maria; Hogaboam, Cory M; Kunkel, Stephen L; Delaney, Stephen; Christie, Mark I; Perretti, Mauro

    2001-01-01

    Chemokine expression and function was monitored in an experimental model of granulomatous tissue formation after injection of croton oil in complete Freund's adjuvant (CO/CFA) into mouse dorsal air-pouches up to 28 days. In the first week, mast cell degranulation and leukocyte influx (mononuclear cell, MNC, and polymorphonuclear cell, PMN) were associated with CXCR2, KC and macrophage inflammatory protein (MIP)-2 mRNA expression, as determined by TaqMan® reverse transcriptase-polymerase chain reaction. KC (∼400 pg mg protein−1, n=12) and MIP-2 (∼800 pg mg protein−1, n=12) proteins peaked at day 7, together with myeloperoxidase (MPO) activity. Highest MIP-1α (>1 ng mg protein−1, n=12) levels were measured at day 3. After day 7, a gradual increase in CCR2 and CCR5 mRNA, monocyte chemoattractant protein (MCP)-1 mRNA and protein expression was measured. MCP-1 protein peaked at day 21 (∼150 pg mg protein−1, n=12) and was predominantly expressed by mast cells. A gradual increase in N-acetyl-β-D-glucosaminidase (NAG) activity (maximal at 28 days) was also measured. An antiserum against MIP-1α did not modify the inflammatory response measured at day 7 (except for a 50% reduction in MIP-1α levels), but provoked a significant increase in MPO, NAG and MCP-1 levels as measured at day 21 (n=6, P<0.05). An antiserum to MCP-1 reduced NAG activity at day 21 but increased MPO activity values (n=8, P<0.05). In conclusion, we have shown that CO/CFA initiates a complex inflammatory reaction in which initial expression of MIP-1α serves a protective role whereas delayed expression of MCP-1 seems to have a genuine pro-inflammatory role. PMID:11704636

  16. Early plasma monocyte chemoattractant protein 1 predicts the development of sepsis in trauma patients: A prospective observational study.

    PubMed

    Wang, Yuchang; Liu, Qinxin; Liu, Tao; Zheng, Qiang; Xu, Xi'e; Liu, Xinghua; Gao, Wei; Li, Zhanfei; Bai, Xiangjun

    2018-04-01

    Monocyte chemoattractant protein 1 (MCP-1) is an initiating cytokine of the inflammatory cascade. Extracellular MCP-1 exhibits pro-inflammatory characteristic and plays a central pathogenic role in critical illness. The purpose of the study was to identify the association between plasma MCP-1 levels and the development of sepsis after severe trauma.The plasma levels of MCP-1 in severe trauma patients were measured by a quantitative enzyme-linked immune sorbent assay and the dynamic release patterns were recorded at three time points during seven days post-trauma. The related factors of prognosis were compared between sepsis and non-sepsis groups and analyzed using multivariate logistic regression analysis. We also used receiver operating characteristic (ROC) curves to assess the values of different variables in predicting sepsis.A total of 72 patients who met criteria indicative of severe trauma (72.22% of male; mean age, 49.40 ± 14.29 years) were enrolled. Plasma MCP-1 concentrations significantly increased on post-trauma day 1 and that this increase was significantly correlated with the Injury Severity Score (ISS) and interleukin-6 (IL-6). Multivariate logistic regression analysis showed that early MCP-1, ISS, and IL-6 were independent risk factors for sepsis in severe trauma patients. Incorporation of the early MCP-1 into the ISS can increase the discriminative performance for predicting development of sepsis.Early plasma MCP-1 concentrations can be used to assess the severity of trauma and is correlated with the development of sepsis after severe trauma. The addition of the early MCP-1 levels to the ISS significantly improves its ability to predict development of sepsis.

  17. Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

    PubMed

    Masuda, Takahiro; Deng, Xue; Tamai, Riyoko

    2009-08-01

    Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

  18. A three-dimensional in vitro model to demonstrate the haptotactic effect of monocyte chemoattractant protein-1 on atherosclerosis-associated monocyte migration

    PubMed Central

    Ghousifam, Neda; Mortazavian, Seyyed Hamid; Bhowmick, Rudra; Vasquez, Yolanda; Blum, Frank D.; Gappa-Fahlenkamp, Heather

    2017-01-01

    Monocyte transendothelial migration is a multi-step process critical for the initiation and development of atherosclerosis. The chemokine monocyte chemoattractant protein-1 (MCP-1) is overexpressed during atheroma and its concentration gradients in the extracellular matrix (ECM) is critical for the transendothelial recruitment of monocytes. Based on prior observations, we hypothesize that both free and bound gradients of MCP-1 within the ECM are involved in directing monocyte migration. The interaction between a three-dimensional (3D), cell-free, collagen matrix and MCP-1; and its effect on monocyte migration was measured in this study. Our results showed such an interaction existed between MCP-1 and collagen, as 26% of the total MCP-1 added to the collagen matrix was bound to the matrix after extensive washes. We also characterized the collagen-MCP-1 interaction using biophysical techniques. The treatment of the collagen matrix with MCP-1 lead to increased monocyte migration, and this phenotype was abrogated by treating the matrix with an anti-MCP-1 antibody. Thus, our results indicate a binding interaction between MCP-1 and the collagen matrix, which could elicit a haptotactic effect on monocyte migration. A better understanding of such mechanisms controlling monocyte migration will help identify target cytokines and lead to the development of better anti-inflammatory therapeutic strategies. PMID:28041913

  19. Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and macrophage inflammatory protein (MIP)-1 beta production by synovial T cells.

    PubMed Central

    Robinson, E; Keystone, E C; Schall, T J; Gillett, N; Fish, E N

    1995-01-01

    Earlier studies from this laboratory provided evidence for restricted cytokine expression in the T cell population in RA tissues. Specifically, IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) gene expression levels were low. The selective chemoattractant and activation effects of chemokines on leucocytes identify them as potentially ideal candidates in mediating selective inflammatory processes in RA. Accordingly, we undertook studies to examine constitutive chemokine gene expression in RA tissues. RANTES, monocyte chemotactic protein-1 (MCP-1) and MIP-1 beta gene expression was examined in both the T and non-T cell populations in RA peripheral blood (PB), synovial fluid (SF) and synovial tissues (ST). Our results identified elevated levels of both RANTES and MIP-1 beta gene expression in circulating RA PB and SF T cells. By contrast, MCP-1 expression was virtually absent in RA PB, yet elevated MCP-1 mRNA levels were detected primarily in the non-T cell populations of the SF and ST samples. Histological examination of affected rheumatoid joints revealed extensive RANTES and MIP-1 beta expression in sites of lymphocyte infiltration and cell proliferation, namely the synovial lining and sublining layers. Fractionation or RA ST patient samples revealed that RANTES expression was restricted to the T cells, whereas MIP-1 beta expression was detected in both T and non-T fractions. These data suggest that MCP-1, MIP-1 beta and RANTES may have a central role in the trafficking of reactive molecules involved in immunoregulation and in the inflammatory processes in RA. Images Fig. 4 PMID:7545093

  20. Monocyte chemoattractant protein-1 and interleukin-8 levels in urine and serum of patents with hemolytic uremic syndrome.

    PubMed

    van Setten, P A; van Hinsbergh, V W; van den Heuvel, L P; Preyers, F; Dijkman, H B; Assmann, K J; van der Velden, T J; Monnens, L A

    1998-06-01

    The epidemic form of the hemolytic uremic syndrome (HUS) in children is hallmarked by endothelial cell damage, most predominantly displayed by the glomerular capillaries. The influx of mononuclear (MO) and polymorphonuclear cells (PMNs) into the glomeruli may be an important event in the initiation, prolongation, and progression of glomerular endothelial cell damage in HUS patients. The molecular mechanisms for the recruitment of these leukocytes into the kidney are unclear, but monocyte chemoattractant protein-1 (MCP-1) and IL-8 are suggested to be prime candidates. In this study, we analyzed the presence of both chemokines in 24-h urinary (n = 15) and serum (n = 14) samples of HUS children by specific ELISAs. Furthermore, kidney biopsies of three different HUS children were examined for MO and PMN cell infiltration by histochemical techniques and electron microscopy. Whereas the chemokines MCP-1 and IL-8 were present in only very limited amounts in urine of 17 normal control subjects, serial samples of HUS patients demonstrated significantly elevated levels of both chemokines. HUS children with anuria showed higher initial and maximum chemokine levels than their counterparts without anuria. A strong positive correlation was observed between urinary MCP-1 and IL-8 levels. Whereas initial serum IL-8 levels were significantly increased in HUS children, serum MCP-1 levels were only slightly elevated compared with serum MCP-1 in control children. No correlation was found between urinary and serum chemokine concentrations. Histologic and EM studies of HUS biopsy specimens clearly showed the presence of MOs and to a lesser extent of PMNs in the glomeruli. The present data suggest an important local role for MOs and PMNs in the process of glomerular endothelial-cell damage. The chemokines MCP-1 and IL-8 may possibly be implicated in the pathogenesis of HUS through the recruitment and activation of MOs and PMNs, respectively.

  1. S100B promotes glioma growth through chemoattraction of myeloid-derived macrophages.

    PubMed

    Wang, Huaqing; Zhang, Leying; Zhang, Ian Y; Chen, Xuebo; Da Fonseca, Anna; Wu, Shihua; Ren, Hui; Badie, Sam; Sadeghi, Sam; Ouyang, Mao; Warden, Charles D; Badie, Behnam

    2013-07-15

    S100B is member of a multigenic family of Ca(2+)-binding proteins, which is overexpressed by gliomas. Recently, we showed that low concentrations of S100B attenuated microglia activation through the induction of Stat3. We hypothesized that overexpression of S100B in gliomas could promote tumor growth by modulating the activity of tumor-associated macrophages (TAM). We stably transfected GL261 glioma cell lines with constructs that overexpressed (S100B(high)) or underexpressed (S100B(low)) S100B and compared their growth characteristics to intracranial wild-type (S100B(wt)) tumors. Downregulation of S100B in gliomas had no impact on cell division in vitro but abrogated tumor growth in vivo. Interestingly, compared to S100B(low) tumors, S100B(wt) and S100B(high) intracranial gliomas exhibited higher infiltration of TAMs, stronger inflammatory cytokine expression, and increased vascularity. To identify the potential mechanisms involved, the expression of the S100B receptor, receptor for advanced glycation end products (RAGE), was evaluated in gliomas. Although S100B expression induced RAGE in vivo, RAGE ablation in mice did not significantly inhibit TAM infiltration into gliomas, suggesting that other pathways were involved in this process. To evaluate other mechanisms responsible for TAM chemoattraction, we then examined chemokine pathways and found that C-C motif ligand 2 (CCL2) was upregulated in S100B(high) tumors. Furthermore, analysis of The Cancer Genome Atlas's glioma data bank showed a positive correlation between S100B and CCL2 expression in human proneural and neural glioma subtypes, supporting our finding. These observations suggest that S100B promotes glioma growth by TAM chemoattraction through upregulation of CCL2 and introduces the potential utility of S100B inhibitors for glioma therapy.

  2. S100B Promotes Glioma Growth through Chemoattraction of Myeloid-Derived Macrophages

    PubMed Central

    Wang, Huaqing; Zhang, Leying; Zhang, Ian Y.; Chen, Xuebo; Da Fonseca, Anna; Wu, Shihua; Ren, Hui; Badie, Sam; Sadeghi, Sam; Ouyang, Mao; Warden, Charles D.; Badie, Behnam

    2013-01-01

    Purpose S100B is member of a multigenic family of Ca2+-binding proteins that is overexpressed by gliomas. Recently, we demonstrated that low concentrations of S100B attenuated microglia activation through the induction of Stat3. We hypothesized that overexpression of S100B in gliomas could promote tumor growth by modulating the activity of tumor-associated macrophages (TAMs). Experimental Design We stably transfected GL261 glioma cell lines with constructs that overexpressed (S100Bhigh) or underexpressed (S100Blow) S100B and compared their growth characteristics to intracranial wild-type (S100Bwt) tumors. Results Downregulation of S100B in gliomas had no impact on cell division in vitro but abrogated tumor growth in vivo. Interestingly, compared to S100Blow tumors, S100Bwt and S100Bhigh intracranial gliomas exhibited higher infiltration of TAMs, stronger inflammatory cytokine expression, and increased vascularity. To identify the potential mechanisms involved, the expression of the S100B receptor, RAGE (receptor for advanced glycation end products), was evaluated in gliomas. Although S100B expression induced RAGE in vivo, RAGE ablation in mice did not significantly inhibit TAM infiltration into gliomas, suggesting that other pathways were involved in this process. To evaluate other mechanisms responsible for TAM chemoattraction, we then examined chemokine pathways and found that CCL2 was upregulated in S100Bhigh tumors. Furthermore, analysis of TCGA’s glioma data bank demonstrated a positive correlation between S100B and CCL2 expression in human proneural and neural glioma subtypes, supporting our finding. Conclusions These observations suggest that S100B promotes glioma growth by TAM chemoattraction through upregulation of CCL2 and introduces the potential utility of S100B inhibitors for glioma therapy. PMID:23719262

  3. Progesterone from the Cumulus Cells Is the Sperm Chemoattractant Secreted by the Rabbit Oocyte Cumulus Complex

    PubMed Central

    Guidobaldi, Héctor Alejandro; Teves, María Eugenia; Uñates, Diego Rafael; Anastasía, Agustín; Giojalas, Laura Cecilia

    2008-01-01

    Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species. PMID:18725941

  4. Early Secreted Antigenic Target of 6 kDa of Mycobacterium tuberculosis Stimulates Macrophage Chemoattractant Protein-1 Production by Macrophages and Its Regulation by p38 Mitogen-Activated Protein Kinases and Interleukin-4.

    PubMed

    Ma, J; Jung, B-G; Yi, N; Samten, B

    2016-07-01

    Early secreted antigenic target of 6 kDa (ESAT-6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT-6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)-1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrow-derived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT-6, but not Ag85A and culture filtrate protein 10 kDa (CFP10), induced MCP-1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen-activated protein kinases (MAPK) and PI3K further enhanced ESAT-6-induced MCP-1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT-6-induced MCP-1 mRNA accumulation without affecting mRNA stability. ESAT-6 also induced TNF-α from BMDMs and MCP-1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine- and cell-specific effect of p38 MAPK inhibition on ESAT-6-induced MCP-1 by macrophages. Pretreatment of BMDMs with IL-4, but not other cytokines (IL-2, IL-10, TNF-α, IFN-γ and IL-1α) further elevated ESAT-6-stimulated MCP-1 production although IL-4 did not induce MCP-1 without ESAT-6. Both p38 MAPK inhibitor and IL-4 did not show additive effect on ESAT-6-induced MCP-1 protein level despite such effect on MCP-1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL-4 in ESAT-6-induced MCP-1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis. © 2016 The Foundation for the Scandinavian Journal of Immunology.

  5. MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype.

    PubMed

    Frangogiannis, Nikolaos G; Mendoza, Leonardo H; Ren, Guofeng; Akrivakis, Spyridon; Jackson, Peggy L; Michael, Lloyd H; Smith, C Wayne; Entman, Mark L

    2003-08-01

    Myocardial infarction is associated with the rapid induction of mononuclear cell chemoattractants that promote monocyte infiltration into the injured area. Monocyte-to-macrophage differentiation and macrophage proliferation allow a long survival of monocytic cells, critical for effective healing of the infarct. In a canine infarction-reperfusion model, newly recruited myeloid leukocytes were markedly augmented during early reperfusion (5-72 h). By 7 days, the number of newly recruited myeloid cells was reduced, and the majority of the inflammatory cells remaining in the infarct were mature macrophages. Macrophage colony-stimulating factor (MCSF) is known to facilitate monocyte survival, monocyte-to-macrophage conversion, and macrophage proliferation. We demonstrated marked induction of MCSF mRNA in ischemic segments persisting for at least 5 days after reperfusion. MCSF expression was predominantly localized to mature macrophages infiltrating the infarcted myocardium; the expression of the MCSF receptor, c-Fms, a protein with tyrosine kinase activity, was found in these macrophages but was also observed in a subset of microvessels within the infarct. Many infarct macrophages expressed proliferating cell nuclear antigen, a marker of proliferative activity. In vitro MCSF induced monocyte chemoattractant protein-1 synthesis in canine venous endothelial cells. MCSF-induced endothelial monocyte chemoattractant protein-1 upregulation was inhibited by herbimycin A, a tyrosine kinase inhibitor, and by LY-294002, a phosphatidylinositol 3'-kinase inhibitor. We suggest that upregulation of MCSF in the infarcted myocardium may have an active role in healing not only through its effects on cells of monocyte/macrophage lineage, but also by regulating endothelial cell chemokine expression.

  6. Interplay between brain stem angiotensins and monocyte chemoattractant protein-1 as a novel mechanism for pressor response after ischemic stroke.

    PubMed

    Chang, Alice Y W; Li, Faith C H; Huang, Chi-Wei; Wu, Julie C C; Dai, Kuang-Yu; Chen, Chang-Han; Li, Shau-Hsuan; Su, Chia-Hao; Wu, Re-Wen

    2014-11-01

    Pressor response after stroke commonly leads to early death or susceptibility to stroke recurrence, and detailed mechanisms are still lacking. We assessed the hypothesis that the renin-angiotensin system contributes to pressor response after stroke by differential modulation of the pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) in the rostral ventrolateral medulla (RVLM), a key brain stem site that maintains blood pressure. We also investigated the beneficial effects of a novel renin inhibitor, aliskiren, against stroke-elicited pressor response. Experiments were performed in male adult Sprague-Dawley rats. Stroke induced by middle cerebral artery occlusion elicited significant pressor response, accompanied by activation of angiotensin II (Ang II)/type I receptor (AT1R) and AT2R signaling, depression of Ang-(1-7)/MasR and Ang IV/AT4R cascade, alongside augmentation of MCP-1/C-C chemokine receptor 2 (CCR2) signaling and neuroinflammation in the RVLM. Stroke-elicited pressor response was significantly blunted by antagonism of AT1R, AT2R or MCP-1/CCR2 signaling, and eliminated by applying Ang-(1-7) or Ang IV into the RVLM. Furthermore, stroke-activated MCP-1/CCR2 signaling was enhanced by AT1R and AT2R activation, and depressed by Ang-(1-7)/MasR and Ang IV/AT4R cascade. Aliskiren inhibited stroke-elicited pressor response via downregulating MCP-1/CCR2 activity and reduced neuroinflammation in the RVLM; these effects were potentiated by Ang-(1-7) or Ang IV. We conclude that whereas Ang II/AT1R or Ang II/AT2R signaling in the brain stem enhances, Ang-(1-7)/MasR or Ang IV/AT4R antagonizes pressor response after stroke by differential modulations of MCP-1 in the RVLM. Furthermore, combined administration of aliskiren and Ang-(1-7) or Ang IV into the brain stem provides more effective amelioration of stroked-induced pressor response. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Enhanced UGT1A1 Gene and Protein Expression in Endometriotic Lesions.

    PubMed

    Piccinato, Carla A; Neme, Rosa M; Torres, Natália; da Silva Victor, Elivane; Brudniewski, Heloísa F; Rosa E Silva, Júlio C; Ferriani, Rui A

    2018-01-01

    The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

  8. Establishment of stable cell line for inducing KAP1 protein expression.

    PubMed

    Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

    2015-06-01

    Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

  9. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain andmore » a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.« less

  10. [Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells].

    PubMed

    Yuan, X L; Li, Y; Pan, X H; Zhou, M; Gao, Q Y; Li, M C

    2016-01-01

    Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

  11. The recombinant expression and activity detection of MAF-1 fusion protein.

    PubMed

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-10-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

  12. The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

    PubMed Central

    Ma, H; Gamper, M; Parent, C; Firtel, R A

    1997-01-01

    We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

  13. The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

    PubMed

    Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

    2002-08-01

    Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

  14. The roles of MCP-1 and protein kinase C delta activation in human eosinophilic leukemia EoL-1 cells.

    PubMed

    Lee, Ji-Sook; Yang, Eun Ju; Kim, In Sik

    2009-12-01

    Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKC delta in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G(i)/G(o) protein, phospholipase C (PLC), PKC delta, p38 MAPK and NF-kappaB. MCP-1 activates p38 MAPK via G(i)/G(o) protein, PLC and PKC delta cascade. MCP-1 also induces NF-kappaB translocation and the activation is inhibited by PKC delta activation. The increase in the basal expression and activity of PKC delta in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKC delta is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKC delta functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

  15. Regulation of RE1 Protein Silencing Transcription Factor (REST) Expression by HIP1 Protein Interactor (HIPPI)*

    PubMed Central

    Datta, Moumita; Bhattacharyya, Nitai P.

    2011-01-01

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

  16. Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

    PubMed

    Datta, Moumita; Bhattacharyya, Nitai P

    2011-09-30

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease.

  17. Blunted activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by geranylgeranylacetone: Involvement of unfolded protein response

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hayakawa, Kunihiro; Hiramatsu, Nobuhiko; Okamura, Maro

    2008-01-04

    Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-{kappa}B and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB{sub 5} subtilase cytotoxin reproduced the suppressive effects of GGA.more » Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.« less

  18. Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

    PubMed

    Bragança, Caio Roberto Soares; Colombo, Lívia Tavares; Roberti, Alvaro Soares; Alvim, Mariana Caroline Tocantins; Cardoso, Silvia Almeida; Reis, Kledna Constancio Portes; de Paula, Sérgio Oliveira; da Silveira, Wendel Batista; Passos, Flavia Maria Lopes

    2015-02-01

    The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

  19. Cross-Desensitization of CCR1, But Not CCR2, Following Activation of the Formyl Peptide Receptor (FPR1)1

    PubMed Central

    Bednar, Filip; Song, Changcheng; Bardi, Giuseppe; Cornwell, William; Rogers, Thomas J.

    2014-01-01

    The cross-regulation of G protein-coupled receptors (GPCRs) plays an important role in the immune response. Studies from several laboratories have suggested that a hierarchy of sensitivities to cross-desensitization exists for the chemoattractant GPCRs. We carried out experiments to study the capacity of the formyl peptide receptor-1 (FPR1) to desensitize chemokine receptors CCR1 and CCR2. Our results show that activation of FPR1 resulted in the desensitization and partial internalization of CCR1, but not CCR2, in both primary human monocytes and HEK293 cells co-expressing CCR1, CCR2, and FPR1 (HR1R2F cells). The desensitization of CCR1 by FPR1 stimulation was not due to the simple depletion of the Ca2+ stores, but was dependent on activation of PKC. Furthermore, we found that the cross-desensitization of CCR1 by FPR1 was associated with CCR1 phosphorylation, and moderate reduction of CCR1 cell surface expression. In contrast, CCR2 was not phosphorylated or internalized following FPR1 activation. Additional studies showed that optimal cross-talk between FPR1 and CCR1 were dependent on the functional activity of PKCβ. These results provide a mechanistic basis for the capacity of certain GPCR ligands to exert rapid and selective cross-inactivation of other chemoattractant receptors, and suggest that FPR1 is able to exert “traffic control” in the migration of inflammatory cells by rapidly inhibiting the cell responses to potentially “low priority” chemoattractants such as CCR1 agonists without inhibiting the response to “higher priority” CCR2 chemoattractants. PMID:24778447

  20. Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

    PubMed

    Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

    2005-06-14

    The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

  1. Expression and in vitro functional analyses of recombinant Gam1 protein

    PubMed Central

    Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2014-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

  2. The expression and function of epithelial membrane protein 1 in laryngeal carcinoma.

    PubMed

    Li, Hong; Zhang, Xiaowen; Jiang, Xuejun; Ji, Xu

    2017-01-01

    In this study, we compared the expression of epithelial membrane protein 1 (EMP1) on the steady-state mRNA level (by quantitative real-time PCR) and on the protein level (by western immunoblot and immunohistochemistry) in 51 pairs of laryngeal carcinoma tissues and matched cancer-free peritumor tissues, and we analyzed the correlation between EMP1 expression and different clinicopathological factors. Furthermore, we ectopically expressed EMP1 in human laryngeal carcinoma Hep-2 cells and examined the effects on cell viability, apoptosis, colonogenicity, and motility, by MTT assay, flow cytometry, colony formation assay and Transwell migration assay, respectively. EMP1 expression (on both the mRNA and protein levels) was significantly lower in the cancer tissues than in matched peritumor tissues (P<0.05). In laryngeal cancers, the level of EMP1 protein was correlated with histological grade (P<0.05), but not with age, gender, clinical stage, cancer subtype or lymph node metastasis (P>0.05). Functionally, ectopic expression of EMP1 in Hep-2 cells significantly reduced cell viability, colony formation, and migration, but enhanced apoptosis. Therefore, EMP1 is a tumor suppressor in laryngeal carcinoma. Boosting EMP1 expression in laryngeal carcinoma initiates multiple anticancer phenotypes and thus presents a promising therapeutic strategy for laryngeal cancer.

  3. The Drosophila Extradenticle and Homothorax selector proteins control branchless/FGF expression in mesodermal bridge-cells.

    PubMed

    Merabet, Samir; Ebner, Andreas; Affolter, Markus

    2005-08-01

    The stereotyped outgrowth of tubular branches of the Drosophila tracheal system is orchestrated by the local and highly dynamic expression profile of branchless (bnl), which encodes a secreted fibroblast growth factor (FGF)-like molecule. Despite the importance of the spatial and temporal bnl regulation, little is known about the upstream mechanisms that establish its complex expression pattern. Here, we show that the Extradenticle and Homothorax selector proteins control bnl transcription in a single cell per segment, the mesodermal bridge-cell. In addition, we observed that a key determinant of bridge-cell specification, the transcription factor Hunchback, is also required for bnl expression. Therefore, we propose that one of the functions of the bridge-cell is to synthesize and secrete the chemoattractant Bnl. These findings provide a hitherto unknown and interesting link between combinatorial inputs of transcription factors, cell-specific ligand expression and organ morphogenesis.

  4. Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

    PubMed Central

    Góes, Luiz Gustavo Bentim; de Freitas, Antonio Carlos; Ferraz, Oilita Pereira; Rieger, Tania Tassinari; dos Santos, José Ferreira; Pereira, Alexandre; Beçak, Willy; Lindsey, Charles J.; de Cassia Stocco, Rita

    2008-01-01

    The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression. PMID:24031166

  5. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

  6. Neutrophil migration under spatially-varying chemoattractant gradient profiles.

    PubMed

    Halilovic, Iris; Wu, Jiandong; Alexander, Murray; Lin, Francis

    2015-01-01

    Chemotaxis plays an important role in biological processes such as cancer metastasis, embryogenesis, wound healing, and immune response. Neutrophils are the frontline defenders against invasion of foreign microorganisms into our bodies. To achieve this important immune function, a neutrophil can sense minute chemoattractant concentration differences across its cell body and effectively migrate toward the chemoattractant source. Furthermore, it has been demonstrated in various studies that neutrophils are highly sensitive to changes in the surrounding chemoattractant environments, suggesting the role of a chemotactic memory for processing the complex spatiotemporal chemical guiding signals. Using a microfluidic device, in the present study we characterized neutrophil migration under spatially varying profiles of interleukine-8 gradients, which consist of three spatially ordered regions of a shallow gradient, a steep gradient and a nearly saturated gradient. This design allowed us to examine how neutrophils migrate under different chemoattractant gradient profiles, and how the migratory response is affected when the cell moves from one gradient profile to another in a single experiment. Our results show robust neutrophil chemotaxis in the shallow and steep gradient, but not the saturated gradient. Furthermore, neutrophils display a transition from chemotaxis to flowtaxis when they migrate across the steep gradient interface, and the relative efficiency of this transition depends on the cell's chemotaxis history. Finally, some neutrophils were observed to adjust their morphology to different gradient profiles.

  7. Expression and in vitro functional analyses of recombinant Gam1 protein.

    PubMed

    Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2015-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Adiponectin, interleukin-6, monocyte chemoattractant protein-1, and regional fat mass during 12-month randomized treatment with metformin and/or oral contraceptives in polycystic ovary syndrome.

    PubMed

    Glintborg, Dorte; Mumm, Hanne; Altinok, Magda Lambaa; Richelsen, Bjørn; Bruun, Jens Meldgaard; Andersen, Marianne

    2014-08-01

    Central obesity in polycystic ovary syndrome (PCOS) is associated with increased inflammatory markers and increased risk for type 2 diabetes. To evaluate if improved body composition during treatment with metformin (M) vs. oral contraceptive pills (OCP) was associated with changes in circulating adiponectin, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. Ninety patients with PCOS were randomized to 12-month treatment with M (2 g/day), M + OCP (150 mg desogestrel + 30 microgram ethinylestradiol) or OCP. Adiponectin, IL-6, MCP-1, whole body DXA scans, and clinical evaluations were performed before and after the intervention period in the 65 study completers. Changes in inflammatory markers and changes in total and regional fat mass estimates. Adiponectin, IL-6, and MCP-1 levels were unchanged during the three types of medical intervention. Treatment with M and M + OCP was superior to OCP regarding decreased regional fat mass. Baseline adiponectin and IL-6 were associated with BMI, waist, and trunk fat mass. Changes in trunk fat were significantly associated with changes in IL-6 and MCP-1 during M + OCP. Long-term treatment with M alone or in combination with OCP was associated with improved body composition compared to OCP, whereas inflammatory markers were unchanged. OCP was not associated with increased inflammatory markers despite a small but significant weight gain.

  9. Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

    PubMed Central

    Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

    2003-01-01

    Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

  10. Selective induction of phospholipase D1 in pathogen-activated human monocytes.

    PubMed

    Locati, M; Riboldi, E; Bonecchi, R; Transidico, P; Bernasconi, S; Haribabu, B; Morris, A J; Mantovani, A; Sozzani, S

    2001-08-15

    Phospholipase D (PLD) activation is part of the complex signalling cascade induced during phagocyte activation. Two PLD isoforms have been cloned, but their role in phagocyte functions is still poorly defined. We report that resting fresh circulating human monocytes expressed PLD1. PLD1 protein expression was rapidly down-regulated during cell culture. Lipopolysaccharide and pathogen-derived agonists (Candida albicans, arabinoside-terminated lipoarabinomannan and Gram-positive bacteria, but not mannose-capped lipoarabinomannan or double-stranded RNA) strongly induced PLD1 expression at both the mRNA and protein levels. Pro-inflammatory cytokines [interleukin (IL)-1beta and tumour necrosis factor alpha] had only a weak effect, whereas immune cytokines (IL-6 and interferon gamma), anti-inflammatory cytokines (IL-13 and IL-10) and chemoattractants (fMet-Leu-Phe and macrophage chemoattractant protein 1) were inactive. None of the agonists tested induced significant changes in the basal expression of PLD2 mRNA. Consistent with PLD1 up-regulation was the observation that PLD enzymic activity was higher in monocytes treated with active-pathogen-derived agonists than in control cells, when stimulated with PMA or with chemotactic agonists (fMet-Leu-Phe and C5a). Thus PLD2 seems to be a constitutive enzyme in circulating monocytes. Conversely, PLD1 is an inducible protein, rapidly regulated during culture conditions and selectively induced during cell activation. Therefore PLD1 might have a relevant role in immune responses against pathogens and in chronic inflammation.

  11. Chemoattractant receptors and lymphocyte egress from extralymphoid tissue: changing requirements during the course of inflammation1

    PubMed Central

    Brown, Meghan N.; Fintushel, Sarah R.; Lee, Michael H.; Jennrich, Silke; Geherin, Skye A.; Hay, John B.; Butcher, Eugene C.; Debes, Gudrun F.

    2012-01-01

    Memory/effector T cells traffic efficiently through extralymphoid tissues, entering from the blood and leaving via the afferent lymph. During inflammation, T cell traffic into the affected tissue dramatically increases; however, the dynamics and mechanisms of T cell exit from inflamed tissues are poorly characterized. Here we show, using both a mouse and a sheep model, that large numbers of lymphocytes leave the chronically inflamed skin. Many T cells capable of producing IFN-γ and IL-17 also entered the draining afferent lymph, demonstrating that memory/effector T cells egress from sites of inflammation. Whereas efficient egress from acutely inflamed skin required lymphocyte-expressed CCR7, chronic inflammation promoted significant CCR7-independent exit as well. Lymphocyte exit at late time points of inflammation was sensitive to pertussis toxin but only partially affected by the drug FTY720, implying the contribution of alternative chemoattractant receptors other than S1P1. Our data show that CCR7 is an important receptor for lymphocyte egress from both resting and inflamed extralymphoid tissues, but that alternative exit receptors come into play during chronic inflammation. PMID:20833836

  12. ER stress triggers MCP-1 expression through SET7/9-induced histone methylation in the kidneys of db/db mice.

    PubMed

    Chen, Jigang; Guo, Yanhong; Zeng, Wei; Huang, Li; Pang, Qi; Nie, Ling; Mu, Jiao; Yuan, Fahuan; Feng, Bing

    2014-04-15

    Epigenetics plays a key role in the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Here, we examined the role of endoplasmic reticulum (ER) stress in histone H3 lysine 4 (H3K4) methyltransferase SET7/9-induced monocyte chemoattractant protein-1 (MCP-1) expression in the kidneys of db/db mice. Our results indicate that the expression of MCP-1 significantly increased in the kidneys of db/db mice in a time-dependent manner. An increased chromatin mark associated with an active gene (H3K4me1) at MCP-1 promoters accompanied this change in expression. The expression of SET7/9 and the recruitment to these promoters were also elevated. SET7/9 gene silencing with small interfering (si) RNAs significantly attenuated the expression of H3K4me1 and MCP-1. Furthermore, expression of signaling regulator glucose-regulated protein 78 (GRP78), a monitor of ER stress, significantly increased in the kidneys of db/db mice. The expression of spliced X-box binding protein 1 (XBP1s), an ER stress-inducible transcription factor, and recruitment to the SET7/9 promoters were also increased. XBP1s gene silencing with siRNAs significantly attenuated the expression of SET7/9, H3K4me1, and MCP-1. The chaperone betaine not only effectively downregulated the GRP78 and XBP1s expression levels but also markedly decreased the SET7/9, H3K4me1, and MCP-1 levels. Luciferase reporter assay demonstrated that XBP1s participated in ER stress-induced SET7/9 transcription, Taken together, these results reveal that ER stress can trigger the expression of MCP-1, in part through the XBP1s-mediated induction of SET7/9.

  13. N-caffeoyltryptomine, a potent anti-inflammatory phenolic amide, suppressed MCP-1 expression in LPS-stimulated THP-1 cells and rats fed with a high fat diet

    USDA-ARS?s Scientific Manuscript database

    Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of cardiovascular diseases such as arthrosclerosis. N-caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential e...

  14. Anabolic Properties of High Mobility Group Box Protein-1 in Human Periodontal Ligament Cells In Vitro

    PubMed Central

    Wolf, Michael; Lossdörfer, Stefan; Römer, Piero; Bastos Craveiro, Rogerio; Deschner, James; Jäger, Andreas

    2014-01-01

    High mobility group box protein-1 (HMGB1) is mainly recognized as a chemoattractant for macrophages in the initial phase of host response to pathogenic stimuli. However, recent findings provide evidence for anabolic properties in terms of enhanced proliferation, migration, and support of wound healing capacity of mesenchymal cells suggesting a dual role of the cytokine in the regulation of immune response and subsequent regenerative processes. Here, we examined potential anabolic effects of HMGB1 on human periodontal ligament (PDL) cells in the regulation of periodontal remodelling, for example, during orthodontic tooth movement. Preconfluent human PDL cells (hPDL) were exposed to HMGB1 protein and the influence on proliferation, migration, osteogenic differentiation, and biomineralization was determined by MTS assay, real time PCR, immunofluorescence cytochemistry, ELISA, and von Kossa staining. HMGB1 protein increased hPDL cell proliferation, migration, osteoblastic marker gene expression, and protein production as well as mineralized nodule formation significantly. The present findings support the dual character of HMGB1 with anabolic therapeutic potential that might support the reestablishment of the structural and functional integrity of the periodontium following periodontal trauma such as orthodontic tooth movement. PMID:25525297

  15. Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

    PubMed

    Pushnova, E A; Ostanin, K; Thelen, M P

    2001-11-01

    Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

  16. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  17. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    PubMed Central

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm. PMID:23826410

  18. Methylation status and protein expression of RASSF1A in breast cancer patients.

    PubMed

    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  19. Long interspersed element-1 protein expression is a hallmark of many human cancers.

    PubMed

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

    2014-05-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  20. Downregulated expression of the cyclase-associated protein 1 (CAP1) reduces migration in esophageal squamous cell carcinoma.

    PubMed

    Li, Mei; Yang, Xiaojing; Shi, Hui; Ren, Hanru; Chen, Xueyu; Zhang, Shu; Zhu, Junya; Zhang, Jianguo

    2013-09-01

    Overexpression of cyclase-associated proteins has been associated with poor prognosis in several human cancers. Cyclase-associated protein 1 is a member of the cyclase-associated proteins which contributes to tumor progression. The aim of the present study was to examine the expression of cyclase-associated protein 1 and to elucidate its clinicopathologic significance in a larger series of esophageal squamous cell carcinoma. Immunohistochemical and western blot analyses were performed in esophageal squamous cell carcinoma tissues. Survival analyses were performed by using the Kaplan-Meier method. The role of cyclase-associated protein 1 in migration was studied in esophageal squamous cell carcinoma cell lines of TE1 through knocking down cyclase-associated protein 1 with siRNA and overexpression of cyclase-associated protein 1. The regulation of cyclase-associated protein 1 on migration was determined by transwell and wound-healing assays. Immunohistochemical analysis showed that cyclase-associated protein 1 expression was negatively associated with E-cadherin and significantly associated with lymph node metastases. Survival analysis revealed that cyclase-associated protein 1 overexpression was significantly associated with overall survival (P = 0.011). Knock down of cyclase-associated protein 1 in TE1 cells resulted in decreased vimentin and F-actin levels and the capability for migration. In addition, overexpression of cyclase-associated protein 1 promoted the migration of TE1 cells. These findings suggest that cyclase-associated protein 1 is involved in the metastasis of esophageal squamous cell carcinoma, and that elevated levels of cyclase-associated protein 1 expression may indicate a poor prognosis for patients with esophageal squamous cell carcinoma.

  1. CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury

    PubMed Central

    Weinreuter, Martin; Kreusser, Michael M; Beckendorf, Jan; Schreiter, Friederike C; Leuschner, Florian; Lehmann, Lorenz H; Hofmann, Kai P; Rostosky, Julia S; Diemert, Nathalie; Xu, Chang; Volz, Hans Christian; Jungmann, Andreas; Nickel, Alexander; Sticht, Carsten; Gretz, Norbert; Maack, Christoph; Schneider, Michael D; Gröne, Hermann-Josef; Müller, Oliver J; Katus, Hugo A; Backs, Johannes

    2014-01-01

    CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKIIδC was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed, nor in cardiomyocyte-specific CaMKIIδ/γ double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1α, MIP-1α). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes. PMID:25193973

  2. The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans.

    PubMed

    Tseng, Rong-Jeng; Armstrong, Kristin R; Wang, Xiaodong; Chamberlin, Helen M

    2007-11-01

    In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences.

  3. Anti-inflammatory effect of resveratrol on TNF-{alpha}-induced MCP-1 expression in adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu Jian; Key Laboratory of Human Functional Genomics of Jiangsu Province, School of Basic Medical Science, Jiangsu Province Diabetes Center, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029; Yong Wei

    2008-05-02

    Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-{alpha}-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-{alpha}-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-{alpha}-induced MCP-1 transcription. Nuclearmore » factor (NF)-{kappa}B was determined to play a major role in the TNF-{alpha}-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-{kappa}B complex and subsequently suppressed NF-{kappa}B transcriptional activity in TNF-{alpha}-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies.« less

  4. The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

    PubMed Central

    Caswell, J L; Middleton, D M; Gordon, J R

    2001-01-01

    Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions. PMID:11768129

  5. Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells.

    PubMed

    Galanakis, Emmanouil; Di Cello, Francescopaolo; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

    2006-12-01

    Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.

  6. Cigarette smoke induces the expression of Notch3, not Notch1, protein in lung adenocarcinoma.

    PubMed

    Cheng, Zhenshun; Tan, Qiuyue; Tan, Weijun; Zhang, L I

    2015-08-01

    The aim of the present study was to determine the effect of cigarette smoke on the expression of Notch proteins in lung adenocarcinoma (LAC). Protein expression levels of Notch1 and Notch3 were analyzed using immunohistochemistry in 102 human LAC specimens. Of these, 52 were obtained from smokers and 50 from non-smokers. In addition, cigarette smoke extract (CSE) at varying concentrations (1, 2.5 and 5%) was administered to A549 cells. The expression of Notch1 and Notch3 protein was then detected by western blot analysis at different time points (0, 8, 24 and 48 h). Of the 102 LAC specimens, 42 (41.2%) were positive for Notch1 and 63 (61.8%) were positive for Notch3. There was no significant difference in the level of Notch1 expression between smokers and non-smokers with LAC (P>0.05). The positive rate and staining intensity of Notch3 expression were increased in the smokers compared with the non-smokers (P<0.05). The expression of Notch3 protein in A549 cells increased in a time- and dose-dependent manner following treatment with CSE, whilst the expression of Notch1 protein appeared stable. The results suggested that cigarette smoke was able to induce the expression of Notch3, not Notch1, protein in LAC. The data revealed an upregulation of Notch3 in LAC following cigarette smoke exposure. Such findings may provide a novel therapeutic target for the treatment of LAC.

  7. IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

    PubMed

    Lara-Diaz, V J; Castilla-Cortazar, I; Martín-Estal, I; García-Magariño, M; Aguirre, G A; Puche, J E; de la Garza, R G; Morales, L A; Muñoz, U

    2017-05-01

    Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1 +/- , and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

  8. Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes.

    PubMed

    Woo, Hae-Mi; Kang, Ji-Hye; Kawada, Teruo; Yoo, Hoon; Sung, Mi-Kyung; Yu, Rina

    2007-02-13

    Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes

  9. [Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

    PubMed

    Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

    2014-06-01

    This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

  10. Protein regulator of cytokinesis-1 expression: prognostic value in lung squamous cell carcinoma patients

    PubMed Central

    Zhan, Ping; Xi, Guang-Min; Liu, Hong-Bing; Liu, Ya-Fang; Xu, Wu-Jian; Zhu, Qingqing; Zhou, Ze-Jun; Miao, Ying-Ying; Wang, Xiao-Xia; Jin, Jia-Jia

    2017-01-01

    Background Protein regulator of cytokinesis-1 (PRC1) has been shown to participate in the completion of cytokinesis, and it is dysregulated in cancer processes. However, its relevance in lung squamous cell carcinoma (SCC) remained largely unknown. We aimed to study the expression pattern of PRC1 and assess its clinical significance in lung SCC. Methods PRC1 protein expression in human lung SCC and adjacent normal lung tissues was detected by immunohistochemistry. PRC1 expression was assessed in association with clinicopathological features and clinical outcomes of lung SCC patients. Results In lung SCC tissues, PRC1 protein expression was significantly higher than those in paired normal lung tissues. The lung SCC patients with PRC1 overexpression had an advanced pathological stage (TNM stage), positive lymph node metastasis, and a shorter overall survival (OS) time more frequently than patients with low PRC1 expression. Additional, PRC1 expression was also shown to be poor as a prognostic factor for OS in patients with lung SCC. Conclusions Our study indicated that aberrant expression of PRC1 may point to biochemical recurrence in lung SCC. This highlights its potential as a valuable prognostic marker for lung SCC. PMID:28840006

  11. Cyanidin 3-glucoside ameliorates hyperglycemia and insulin sensitivity due to downregulation of retinol binding protein 4 expression in diabetic mice.

    PubMed

    Sasaki, Rie; Nishimura, Natsumi; Hoshino, Hiromi; Isa, Yasuka; Kadowaki, Maho; Ichi, Takahito; Tanaka, Akihito; Nishiumi, Shin; Fukuda, Itsuko; Ashida, Hitoshi; Horio, Fumihiko; Tsuda, Takanori

    2007-12-03

    Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine expression is one of the most important targets for the prevention of obesity and improvement of insulin sensitivity. In this study, we have demonstrated that anthocyanin (cyanidin 3-glucoside; C3G) which is a pigment widespread in the plant kingdom, ameliorates hyperglycemia and insulin sensitivity due to the reduction of retinol binding protein 4 (RBP4) expression in type 2 diabetic mice. KK-A(y) mice were fed control or control +0.2% of a C3G diet for 5 weeks. Dietary C3G significantly reduced blood glucose concentration and enhanced insulin sensitivity. The adiponectin and its receptors expression were not responsible for this amelioration. C3G significantly upregulated the glucose transporter 4 (Glut4) and downregulated RBP4 in the white adipose tissue, which is accompanied by downregulation of the inflammatory adipocytokines (monocyte chemoattractant protein-1 and tumor necrosis factor-alpha) in the white adipose tissue of the C3G group. These findings indicate that C3G has significant potency in an anti-diabetic effect through the regulation of Glut4-RBP4 system and the related inflammatory adipocytokines.

  12. Age-associated decrease in GDNF and its cognate receptor GFRα-1 protein expression in human skin.

    PubMed

    Adly, Mohamed A; Assaf, Hanan A; Hussein, Mahmoud Rezk Abdelwahed

    2016-06-01

    Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are expressed in normal human skin. They are involved in murine hair follicle morphogenesis and cycling control. We hypothesize that 'GDNF and GFRα-1 protein expression in human skin undergoes age-associated alterations. To test our hypothesis, the expression of these proteins was examined in human skin specimens obtained from 30 healthy individuals representing three age groups: children (5-18 years), adults (19-60 years) and the elderly (61-81 years). Immunofluorescent and light microscopic immunohistologic analyses were performed using tyramide signal amplification and avidin-biotin complex staining methods respectively. GDNF mRNA expression was examined by RT-PCR analysis. GDNF mRNA and protein as well as GFRα-1 protein expressions were detected in normal human skin. We found significantly reduced epidermal expression of these proteins with ageing. In the epidermis, the expression was strong in the skin of children and declined gradually with ageing, being moderate in adults and weak in the elderly. In children and adults, the expression of both GDNF and GFRα-1 proteins was strongest in the stratum basale and decreased gradually towards the surface layers where it was completely absent in the stratum corneum. In the elderly, GDNF and GFRα-1 protein expression was confined to the stratum basale. In the dermis, both GDNF and GFRα-1 proteins had strong expressions in the fibroblasts, sweat glands, sebaceous glands, hair follicles and blood vessels regardless of the age. Thus there is a decrease in epidermal GDNF and GFRα-1 protein expression in normal human skin with ageing. Our findings suggest that the consequences of this is that GFRα-1-mediated signalling is altered during the ageing process. The clinical and therapeutic ramifications of these observations mandate further investigations. © 2016 The Authors. International Journal of Experimental Pathology © 2016

  13. Characterization of the Expression of the Petunia Glycine-Rich Protein-1 Gene Product 1

    PubMed Central

    Condit, Carol M.; McLean, B. Gail; Meagher, Richard B.

    1990-01-01

    We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16667509

  14. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

  15. Folic acid is a potent chemoattractant of free-living amoebae in a new and amazing species of protist, Vahlkampfia sp.

    PubMed

    Maeda, Yasuo; Mayanagi, Taira; Amagai, Aiko

    2009-03-01

    Folic acid (folate; vitamin Bc) is well recognized as essential for the proper metabolism of the essential amino acid methionine as well as for the synthesis of adenine and thymine. A folate deficiency has been Implicated in a wide variety of disorders from Alzheimer's disease to depression and neural tube defects. In the cellular slime molds, including Dictyostelium, vegetative growth-phase cells are known to chemotactically move toward folate that is secreted by bacterial food sources such as Escherichia coli. Intracellular folate signal transductlon, including G proteins, Ca(2+)channels, and the PIP3 pathway, has been reported in D. discoideum. To our surprise, the genuine chemoattractant(s) of free-living protozoan amoebae have remained to be determined, possibly because of lack of a pertinent method for assaying chemotaxis. We recently isolated a primitive free-living amoeba from the soil of Costa Rica and identified it as a new species of the genus Vahlkampfia belonging to Subclass Gymnamoebia, which includes Entamoeba and Acanthamoeba. The amoebae can grow and multiply quite rapidly, engulfing nearby bacteria such as E. coli. Importantly, we have demonstrated here using a quite simple but finely designed chemotaxis assay that the Vahlkampfia amoebae exhibit chemotaxis toward higher folate concentrations. Riboflavin and cyanocobalamin were also found to serve as positive chemoattractants. Among these chemoattractants, folate is of particular importance because its function seems to be evolutionarily conserved as a potent chemoattractant of amoeboid cells in a wide range of organisms as well as in the Protista and cellular slime molds.

  16. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16more » E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.« less

  17. Estrogen decreases tight junction protein ZO-1 expression in human primary gut tissues.

    PubMed

    Zhou, Zejun; Zhang, Lumin; Ding, Miao; Luo, Zhenwu; Yuan, Shao; Bansal, Meena B; Gilkeson, Gary; Lang, Ren; Jiang, Wei

    2017-10-01

    Females have a higher prevalence of most autoimmune diseases; however, the mechanism is unknown. In this study, we examined the expression of tight junction protein zonula occludens 1 (ZO-1) and estrogen receptor (ER)-α/β in human primary gut tissues by immunohistochemistry, immunofluorescence and qPCR. The expression of ZO-1 and ER-β but not ER-α was present in both male and female gut tissues. There was no sex difference in ER-β expression, but ZO-1 expression was decreased in females compared to males. In vitro, estrogen treatment decreased ZO-1 mRNA and protein expression, ZO-1 promoter activity, IL-6 production, and NF-κB activation in human primary gut tissues or the Caco-2 cells, but increased the ER-β expression in Caco-2 cells. Consistently, plasma IL-6 levels in females were reduced relative to males in vivo. Our finding indicates that estrogen may play a role in gut tight junction expression and permeability. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Heat shock protein 70 regulates Tcl1 expression in leukemia and lymphomas

    PubMed Central

    Gaudio, Eugenio; Paduano, Francesco; Ngankeu, Apollinaire; Lovat, Francesca; Fabbri, Muller; Sun, Hui-Lung; Gasparini, Pierluigi; Efanov, Alexey; Peng, Yong; Zanesi, Nicola; Shuaib, Mohammed A.; Rassenti, Laura Z.; Kipps, Thomas J.; Li, Chenglong; Aqeilan, Rami I.; Lesinski, Gregory B.; Trapasso, Francesco

    2013-01-01

    T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The inhibition of Hsp70 significantly reduced the growth of lymphoma xenografts in vivo and down-regulated the expression of Tcl1 protein. Our findings reveal a functional interaction between Tcl1 and Hsp70 and identify Tcl1 as a novel Hsp70 client protein. These findings suggest that inhibition of Hsp70 may represent an alternative effective therapy for chronic lymphocytic leukemia and lymphomas via its ability to inhibit the oncogenic functions of Tcl1. PMID:23160471

  19. Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana

    PubMed Central

    Cordoba, Elizabeth; Aceves-Zamudio, Denise Lizeth; Hernández-Bernal, Alma Fabiola; Ramos-Vega, Maricela; León, Patricia

    2015-01-01

    Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. PMID:25281700

  20. Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

    PubMed

    Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

    2016-09-01

    Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

    PubMed

    Chalick, Michael; Jacobi, Oded; Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B; Wreschner, Daniel H

    2016-01-01

    Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions

  2. MUC1-ARF—A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA

    PubMed Central

    Pichinuk, Edward; Garbar, Christian; Bensussan, Armand; Meeker, Alan; Ziv, Ravit; Zehavi, Tania; Smorodinsky, Nechama I.; Hilkens, John; Hanisch, Franz-Georg; Rubinstein, Daniel B.; Wreschner, Daniel H.

    2016-01-01

    Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent’ protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its ‘parent’ MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions

  3. Gestational Protein Restriction Reduces Expression of Hsd17b2 in Rat Placental Labyrinth1

    PubMed Central

    Gao, Haijun; Yallampalli, Uma; Yallampalli, Chandra

    2012-01-01

    ABSTRACT Accumulating evidence strongly supports the premise that testosterone may be a key player in fetal programming on hypertension. Studies have shown that gestational protein restriction doubles the plasma testosterone levels in pregnant rats. In this study, we hypothesized that elevated testosterone levels in response to gestational protein restriction were caused by enhanced expression of steroidogenic enzymes or impaired expression of Hsd17b2, a known testosterone inactivator that converts testosterone to androstenedione in placenta. Pregnant Sprague-Dawley rats were fed normal (20% protein, control; n = 10) or a low-protein diet (6% protein, PR; n = 10) from Day 1 of pregnancy until killed at Days 14, 18, or 21. Junctional (JZ) and labyrinth (LZ) zones of placenta were collected for expression assay on steroidogenic genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b2, and Srd5a1) by real-time PCR. The main findings include the following: 1) expressions of Cyp11a1, Hsd3b1, and Cyp17a1 in JZ were not affected by diet but were affected by day of pregnancy; 2) expression of Hsd17b2 in both female and male JZs was remarkably increased by PR at Days 18 and 21 of pregnancy; 3) expressions of Hsd17b2 were reduced by PR in both female and male LZ at Day 18 of pregnancy and in female LZ at Day 21 of pregnancy; and 4) expression of Srd5a1in LZ was not affected by day of pregnancy, gender, or diet. These results indicate that in response to gestational protein restriction, Hsd17b2 may be a key regulator of testosterone levels and associated activities in placental zones, apparently in a paradoxical manner. PMID:22837477

  4. The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

    PubMed Central

    Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

    2012-01-01

    Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

  5. [Expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain].

    PubMed

    Zhang, Wei; Fan, Li-mei; Li, Lin-lin; Peng, Zheng-yu

    2014-01-01

    To investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain. Brain samples were collected from mice with different developmental stages: 9, 12, 14 d before birth (E9, E12, E14) and 1 d, 3 weeks and 3 months after birth. The expression of NSSR1 in mouse brain at different developmental stages was detected by Western blot and the distribution of NSSR1 was analyzed by immunohistochemical staining. The expression and distribution of NSSR1 in mouse brain were compared among embryos, neonatal and adult animals. During embryogenesis, the expression of NSSR1 proteins increases significantly from 0.186(E9) to 0.445(E14) and reached a high level after birth. Immunohistochemical analysis showed that in E12 embryos, NSSR1 was specifically distributed in the marginal and mantle layers. The expression of NSSR1 in hippocampus was very low in neonatal animals but stronger in adults. In cerebellar cortex, NSSR1 was widely expressed in purkinje and granule cells of adult animals, but mainly expressed in Purkinje cells in neonates. The expression of NSSR1 is regulated by the development of mouse brain and presents dynamic changes.

  6. Magnolol affects expression of IGF-1 and associated binding proteins in human prostate cancer cells in vitro.

    PubMed

    McKeown, Brendan T; Hurta, Robert A R

    2014-11-01

    This study investigated the effects of magnolol, a compound from Magnolia officinalis, on the behavior of LNCaP and PC3 human prostate cancer cells in vitro. In vitro cell culture approach with biochemical tests and Western blot analyses was used. Magnolol, (80 μM, 6 hour exposure) was found to affect the expression of insulin-like growth factor-1 (IGF-1) and associated proteins. In both cell lines, protein expression of IGF-1 and insulin-like growth factor binding protein-5 (IGFBP-5) were significantly decreased, while protein expression of IGFBP-3 was significantly increased. Additionally, protein expression of insulin-like growth factor-1 receptor (IGF-1R) was significantly increased and the phosphorylated form of IGF-1 (p-IGF-1R) was significantly decreased in PC3 cells, while IGFBP-4 protein expression was significantly increased in LNCaP cells. This study has demonstrated for the first time that magnolol can alter the expression of IGF-1 and associated proteins in human prostate cancer cells in vitro and suggests that magnolol may have a potential role as a novel anti-prostate cancer agent. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

    PubMed

    Aloulou, Ahmed; Grandval, Philippe; De Caro, Josiane; De Caro, Alain; Carrière, Frédéric

    2006-06-01

    High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.

  8. SIRT1 Gene Polymorphisms Affect the Protein Expression in Cardiovascular Diseases

    PubMed Central

    Kilic, Ulkan; Gok, Ozlem; Bacaksiz, Ahmet; Izmirli, Muzeyyen; Elibol-Can, Birsen; Uysal, Omer

    2014-01-01

    Cardiovascular disease (CVD), the leading cause of death worldwide, is related to gene-environment interactions due to epigenetic factors. SIRT1 protein and its downstream pathways are critical for both normal homeostasis and protection from CVD-induced defects. The aim of this study was to investigate the association between SIRT1 single nucleotide polymorphisms (SNPs) (rs7895833 A>G in the promoter region, rs7069102 C>G in intron 4 and rs2273773 C>T in exon 5 silent mutation) and SIRT1 and eNOS (endothelial nitric oxide synthase) protein expression as well as total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) in CVD patients as compared to controls. The frequencies of mutant genotypes and alleles for rs7069102 and rs2273773 were significantly higher in patients with CVD compared to control group. The risk for CVD was increased by 2.4 times for rs7069102 and 1.9 times for rs2273773 in carriers of mutant allele compared with carriers of wild-type allele pointing the protective role of C allele for both SNPs against CVD. For rs7895833, there was no significant difference in genotype and allele distributions between groups. SIRT1 protein, TAS, TOS and OSI levels significantly increased in patients as compared to control group. In contrast, level of eNOS protein was considerably low in the CVD patients. An increase in the SIRT1 expression in the CVD patients carrying mutant genotype for rs7069102 and heterozygote genotype for all three SNPs was observed. This is the first study reporting an association between SIRT1 gene polymorphisms and the levels of SIRT1 and eNOS expressions as well as TAS, TOS and OSI. PMID:24587358

  9. Statins Inhibit Monocyte Chemotactic Protein 1 Expression in Endometriosis

    PubMed Central

    Cakmak, Hakan; Basar, Murat; Seval-Celik, Yasemin; Osteen, Kevin G.; Duleba, Antoni J.; Taylor, Hugh S.; Lockwood, Charles J.; Arici, Aydin

    2012-01-01

    Statins are potent inhibitors of the endogenous mevalonate pathway. Besides inhibiting cholesterol biosynthesis, statins may also demonstrate anti-inflammatory properties. Inflammation is implicated in the attachment and invasion of endometrial cells to the peritoneal surface and growth of ectopic endometrium by inducing proliferation and angiogenesis. In this study, the effect of statins on monocyte chemotactic protein 1 (MCP-1) expression in endometriotic implants in nude mouse model and in cultured endometriotic cells was evaluated. In mouse model, simvastatin decreased MCP-1 expression in a dose-dependent manner in endometriotic implants (P < .05). Similarly, both simvastatin and mevastatin revealed a dose-dependent inhibition of MCP-1 production in cultured endometriotic cells (P < .01). This inhibitory effect of the statins on MCP-1 production was reversed by the downstream substrates of the mevalonate pathway. Moreover, statins decreased MCP-1 messenger RNA expression in cultured endometriotic cells (P < .05). In conclusion, statins exert anti-inflammatory effect in endometriotic cells and could provide a potential treatment of endometriosis in the future. PMID:22267540

  10. The group migration of Dictyostelium cells is regulated by extracellular chemoattractant degradation.

    PubMed

    Garcia, Gene L; Rericha, Erin C; Heger, Christopher D; Goldsmith, Paul K; Parent, Carole A

    2009-07-01

    Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA(-) cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA(-) cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact.

  11. The Group Migration of Dictyostelium Cells Is Regulated by Extracellular Chemoattractant Degradation

    PubMed Central

    Garcia, Gene L.; Rericha, Erin C.; Heger, Christopher D.; Goldsmith, Paul K.

    2009-01-01

    Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA− cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA− cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact. PMID:19477920

  12. Relationship between epicardial adipose tissue adipocyte size and MCP-1 expression.

    PubMed

    Eiras, Sonia; Teijeira-Fernández, Elvis; Salgado-Somoza, Antonio; Couso, Elena; García-Caballero, Tomás; Sierra, Juan; Juanatey, José Ramón González

    2010-08-01

    Adipocyte size has been associated to increase in inflammatory cytokines expression that can be related to the cardiovascular risk of obesity. Epicardial adipose tissue (EAT) was discovered to play a key role in cardiovascular diseases by producing several inflammatory adipokines. We sought to study whether EAT and subcutaneous adipose tissue (SAT) mean adipocyte sizes are related to the expression of adipokines in patients with cardiovascular diseases. We collected EAT, SAT and blood samples from 22 patients aged 70.9 (s.d. 10.3) undergoing heart surgery. Monocyte chemoattractant protein (MCP)-1, interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha were analyzed by real time RT-PCR, ELISA or immunohistochemistry. Hematoxylin-eosin staining was used for adipocyte area calculations. Adipocyte size is negatively correlated to MCP-1 expression (r=-0.475; p=0.034) in EAT and positively correlated in SAT (r=0.438; p=0.047). These trends persisted after stratification for sex and coronary artery disease (CAD), but only the relationship between EAT MCP-1 and adipocyte size reached statistical significance in the larger group of men with CAD. We have observed that SAT adipocyte size is correlated to BMI (r=0.601; p=0.003); whereas only a non-statistically significant trend was observed in EAT. IL-10 and TNF-alpha expression were not associated to adipocyte size in EAT nor SAT. Secondarily, we found that EAT IL-10 expression is higher in patients with CAD. These results suggest that adipocyte size is a negative determinant of MCP-1 expression in EAT and a positive determinant in SAT. These data might partly explain the different implications of EAT and SAT in cardiovascular diseases. Copyright 2010 Elsevier Ltd. All rights reserved.

  13. Expression, Localization of SUMO-1, and Analyses of Potential SUMOylated Proteins in Bubalus bubalis Spermatozoa

    PubMed Central

    Brohi, Rahim Dad; Wang, Li; Hassine, Najla Ben; Cao, Jing; Talpur, Hira Sajjad; Wu, Di; Huang, Chun-Jie; Rehman, Zia-Ur; Bhattarai, Dinesh; Huo, Li-Jun

    2017-01-01

    Mature spermatozoa have highly condensed DNA that is essentially silent both transcriptionally and translationally. Therefore, post translational modifications are very important for regulating sperm motility, morphology, and for male fertility in general. Protein sumoylation was recently demonstrated in human and rodent spermatozoa, with potential consequences for sperm motility and DNA integrity. We examined the expression and localization of small ubiquitin-related modifier-1 (SUMO-1) in the sperm of water buffalo (Bubalus bubalis) using immunofluorescence analysis. We confirmed the expression of SUMO-1 in the acrosome. We further found that SUMO-1 was lost if the acrosome reaction was induced by calcium ionophore A23187. Proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were identified, including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets. The expression of SUMO-1 in the acrosome region of water buffalo sperm and the identification of potentially SUMOylated proteins important for sperm function implicates sumoylation as a crucial PTM related to sperm function. PMID:28659810

  14. Cigarette Smoke-Related Hydroquinone Dysregulates MCP-1, VEGF and PEDF Expression in Retinal Pigment Epithelium in Vitro and in Vivo

    PubMed Central

    Pons, Marianne; Marin-Castaño, Maria E.

    2011-01-01

    Background Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice. Principal Findings MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo. Conclusion We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might

  15. Recombinant VP1 protein of duck hepatitis virus 1 expressed in Pichia pastoris and its immunogenicity in ducks.

    PubMed

    Wang, C; Li, X K; Wu, T C; Wang, Y; Zhang, C J; Cheng, X C; Chen, P Y

    2014-01-01

    The VP1 gene of duck hepatitis virus type 1 (DHV-1) strain VJ09 was amplified by reverse transcription PCR from the liver of a duckling with clinical symptoms of viral hepatitis. The resulting VP1 cDNA was 720 bp in length and encoded a 240-amino-acid protein. In VP1 gene-based phylogenetic analysis, the VJ09 strain grouped with DHV-1 genotype C. The VP1 gene was inserted into the expression vector pPICZαA and expressed in Pichia pastoris. The expressed VP1 protein was purified and identified by western blot analysis. To evaluate the recombinant VP1's immunogenic potential in ducklings, the antibodies raised in the immunized ducklings were titrated by ELISA, and lymphocyte proliferation and virus neutralization assays were performed. The results show that the recombinant VP1 protein induced a significant immune response in ducklings and this could be a candidate for the development of a subunit vaccine against DHV-1 genotype C.

  16. HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

    PubMed

    Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

    2015-02-01

    The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.

    PubMed

    Yi, Bing; Ozerova, Maria; Zhang, Guan-Xin; Yan, Guijun; Huang, Shengdong; Sun, Jianxin

    2015-10-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at post-transcriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium. To elucidate the molecular basis of tumor necrosis factor (TNF)-α-mediated eNOS mRNA instability, biotinylated eNOS 3'-untranslational region (UTR) was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified 2 cytosolic proteins, with molecular weight of 52 and 57 kDa, which specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). RNA gel mobility shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human ECs. In HUVECs, we show that TNF-α markedly increased PTB1 expression, whereas adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium-dependent relaxation. Furthermore, knockdown of PTB1 substantially attenuated TNF-α-induced destabilization of eNOS transcript and downregulation of eNOS expression. These results indicate that PTB1 is essential for regulating eNOS expression at post-transcriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction. © 2015 American Heart Association, Inc.

  18. In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

    PubMed

    Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

    2000-02-01

    Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

  19. Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cAMP-dependent protein kinase

    PubMed Central

    Huang, Holly S.; Turner, David L.; Thompson, Robert C.; Uhler, Michael D.

    2011-01-01

    cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβprotein. PKIβ played a very specific role in the Ascl1-mediated differentiation process since other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells. PMID:21623794

  20. Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

    PubMed

    Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

    2017-05-01

    Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p < 0.0001). Sterol regulatory element binding protein-1 gene expression was positively correlated with body mass index (r = 0.017, p = 0.921) and waist-hip ratio (r = 0.023, p = 0.544) in polycystic ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression

  1. Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

    PubMed

    Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

    2016-08-01

    Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein

  2. Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3.

    PubMed

    Taylor, Karen; Clarke, David J; McCullough, Bryan; Chin, Wutharath; Seo, Emily; Yang, De; Oppenheim, Joost; Uhrin, Dusan; Govan, John R W; Campopiano, Dominic J; MacMillan, Derek; Barran, Perdita; Dorin, Julia R

    2008-03-14

    beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.

  3. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    PubMed

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  4. Expression, purification, characterization and subcellular localization of the goose parvovirus rep1 protein.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Peng, Gaojing; Liu, Guangqing

    2013-07-01

    The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl β-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.

  5. Increase in peripheral blood mononuclear cell Toll-like receptor 2/3 expression and reactivity to their ligands in a cohort of patients with wet age-related macular degeneration

    PubMed Central

    Zhu, Yi; Liang, Liang; Qian, Dan; Yu, Hongsong; Yang, Peizeng; Lei, Bo

    2013-01-01

    Purpose To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD). Methods Blood samples were collected from 25 patients with wet AMD and 25 age-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient centrifugation. Expression of TLR1 to TLR10 mRNAs in PBMCs from 15 patients with wet AMD and 15 controls was assessed with real-time PCR. TLR2 and TLR3 protein levels in PBMCs from six patients with wet AMD and six controls were measured with flow cytometry. After PBMCs were stimulated with peptidoglycan (PGN) and poly(I:C), the specific ligands of TLR2 and TLR3, cytokines interleukin-6 (IL-6), IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) production in 11 patients with wet AMD and 11 controls were assessed. Results TLR2 and TLR3 mRNA and protein expression in the PBMCs of the patients with wet AMD was significantly higher than that in the controls. However, the difference in TLR1 and TLR4–10 mRNA expression between the two groups was not significant. The PBMCs of the patients with wet AMD produced more IL-6 and IL-8 proteins than the controls in response to PGN, a ligand for TLR2, and more IL-6 protein than the controls in response to poly(I:C), the ligand for TLR3. However, there was no significant difference in vascular endothelial growth factor and monocyte chemoattractant protein-1 production between the wet AMD group and the control group when the PBMCs were stimulated with PGN or poly(I:C). Conclusions Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD. PMID:23946637

  6. Homer1a protein expression in schizophrenia, bipolar disorder, and major depression.

    PubMed

    Leber, Stefan L; Llenos, Ida C; Miller, Christine L; Dulay, Jeannette R; Haybaeck, Johannes; Weis, Serge

    2017-10-01

    In recent years, there was growing interest in postsynaptic density proteins in the central nervous system. Of the most important candidates of this specialized region are proteins belonging to the Homer protein family. This family of scaffolding proteins is suspected to participate in the pathogenesis of a variety of diseases. The present study aims to compare Homer1a expression in the hippocampus and cingulate gyrus of patients with major psychiatric disorders including schizophrenia, bipolar disorder and major depression. Immunohistochemistry was used to analyze changes of Homer1a protein expression in the hippocampal formation and the cingulate gyrus from the respective disease groups. Glial cells of the cingulate gyrus gray matter showed decreased Homer1a levels in bipolar disorder when compared to controls. The same results were seen when comparing cingulate gyrus gray matter glial cells in bipolar disorder with major depression. Stratum oriens glial cells of the hippocampus showed decreased Homer1a levels in bipolar disorder when compared to controls and major depression. Stratum lacunosum glial cells showed decreased Homer1a levels in bipolar disorder when compared to major depression. In stratum oriens interneurons Homer1a levels were increased in all disease groups when compared to controls. Stratum lucidum axons showed decreased Homer1a levels in bipolar disorder when compared to controls. Our data demonstrate altered Homer1a levels in specific brain regions and cell types of patients suffering from schizophrenia, bipolar disorder and major depression. These findings support the role of Homer proteins as interesting candidates in neuropsychiatric pathophysiology and treatment.

  7. Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

    PubMed

    Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

    2016-02-01

    Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes

  8. Increased Expression of Stress Inducible Protein 1 in Glioma-Associated Microglia/Macrophages

    PubMed Central

    da Fonseca, Anna Carolina Carvalho; Wang, Huaqing; Fan, Haitao; Chen, Xuebo; Zhang, Ian; Zhang, Leying; Lima, Flavia Regina Souza; Badie, Behnam

    2014-01-01

    Factors released by glioma-associated microglia/macrophages (GAMs) play an important role in the growth and infiltration of tumors. We have previously demonstrated that the co-chaperone stress-inducible protein 1 (STI1) secreted by microglia promotes proliferation and migration of human glioblastoma (GBM) cell lines in vitro. In the present study, in order to investigate the role of STI1 in a physiological context, we used a glioma model to evaluate STI1 expression in vivo. Here, we demonstrate that STI1 expression in both the tumor and in the infiltrating GAMs and lymphocytes significantly increased with tumor progression. Interestingly, high expression of STI1 was observed in macrophages and lymphocytes that infiltrated brain tumors, whereas STI1 expression in the circulating blood monocytes and lymphocytes remained unchanged. Our results correlate, for the first time, the expression of STI1 and glioma progression, and suggest that STI1 expression in GAMs and infiltrating lymphocytes is modulated by the brain tumor microenvironment. PMID:25042352

  9. Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

    NASA Astrophysics Data System (ADS)

    Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

    2013-11-01

    Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

  10. Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)*

    PubMed Central

    Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A.; Schoenhard, Grant; Zemelman, Boris V.; Mechref, Yehia; Raab-Graham, Kimberly F.

    2016-01-01

    Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and

  11. Floor plate chemoattracts crossed axons and chemorepels uncrossed axons in the vertebrate brain.

    PubMed

    Tamada, A; Shirasaki, R; Murakami, F

    1995-05-01

    In the bilaterally symmetrical vertebrate CNS, all developing axons must choose between remaining on the same side of the midline or growing across it. The mechanism underlying this axonal pathfinding is, however, poorly understood. Here we demonstrate that the ventral midline floor plate (FP) chemorepels two types of ipsilaterally projecting axons, one from the alar plate and another from the basal plate in the mesencephalon. We further demonstrate that the FP chemoattracts contralaterally projecting myelencephalic as well as metencephalic axons. The FP at all axial levels displayed both chemoattractive and chemorepellent activities, suggesting that FP chemoattraction and chemorepulsion may be at work throughout the neuraxis. Chemotropic guidance by the FP may therefore play a key role in the establishment of neuronal projection laterality.

  12. Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes.

    PubMed

    Ren, Gang; Eskandari, Parisa; Wang, Siqian; Smas, Cynthia M

    2016-01-15

    The gene for Small Adipocyte Factor 1, Smaf1 (also known as adipogenin, ADIG), encodes a ∼600 base transcript that is highly upregulated during 3T3-L1 in vitro adipogenesis and markedly enriched in adipose tissues. Based on the lack of an obvious open reading frame in the Smaf1 transcript, it is not known if the Smaf1 gene is protein coding or non-coding RNA. Using a peptide from a putative open reading frame of Smaf1 as antigen, we generated antibodies for western analysis. Our studies prove that Smaf1 encodes an adipose-enriched protein which in western blot analysis migrates at ∼10 kDa. Rapid induction of Smaf1 protein occurs during in vitro adipogenesis and its expression in 3T3-L1 adipocytes is positively regulated by insulin and glucose. Moreover, siRNA studies reveal that expression of Smaf1 in adipocytes is wholly dependent on PPARγ. On the other hand, use of siRNA for Smaf1 to nearly abolish its protein expression in adipocytes revealed that Smaf1 does not have a major role in adipocyte triglyceride accumulation, lipolysis or insulin-stimulated pAkt induction. However, immunolocalization studies using HA-tagged Smaf1 reveal enrichment at adipocyte lipid droplets. Together our findings show that Smaf1 is a novel small protein endogenous to adipocytes and that Smaf1 expression is closely tied to PPARγ-mediated signals and the adipocyte phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Synephrine inhibits eotaxin-1 expression via the STAT6 signaling pathway.

    PubMed

    Roh, Kyung-Baeg; Kim, Il-Hyun; Kim, Young-Soo; Lee, Myungjae; Lee, Jung-A; Jung, Eunsun; Park, Deokhoon

    2014-08-08

    Citrus contain various flavonoids and alkaloids that have multiple biological activities. It is known that the immature Citrus contains larger amounts of bioactive components, than do the mature plants. Although Citrus flavonoids are well known for their biological activities, Citrus alkaloids have not previously been assessed. In this study, we identified synephrine alkaloids as an active compound from immature Citrus unshiu, and investigated the effect of synephrine on eotaxin-1 expression. Eotaxin-1 is a potent chemoattractant for eosinophils, and a critical mediator, during the development of eosinophilic inflammation. We found that synephrine significantly inhibited IL-4-induced eotaxin-1 expression. This synephrine effect was mediated through the inhibition of STAT6 phosphorylation in JAK/STAT signaling. We also found that eosinophil recruitment induced by eotaxin-1 overexpression was inhibited by synephrine. Taken together, these findings indicate that inhibiting IL-4-induced eotaxin-1 expression by synephrine occurs primarily through the suppression of eosinophil recruitment, which is mediated by inhibiting STAT6 phosphorylation.

  14. Expression of the Nrf2 and Keap1 proteins and their clinical significance in osteosarcoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Jihong, E-mail: zhangjihong63@163.com; Wang, Xiaojuan, E-mail: yangjian142@163.com; Wu, Wuzhou, E-mail: jiangchunli68@163.com

    Objective: To investigate the expression and clinical significance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in osteosarcoma tissue. Methods: The data of 102 osteosarcoma patients who underwent surgical treatment at our hospital from June 2000 to March 2009 were collected. The expression levels of the Nrf2 and Keap1 proteins in osteosarcoma tissue and normal peritumour tissues were detected by immunohistochemistry, and the relationship between the expression level and the clinical and pathological features as well as the prognosis was explored. Results: The nuclear expression rate of Nrf2 was 77.5% in osteosarcoma tissue, which wasmore » significantly higher than the rate in normal peritumour bone tissue (9.8%) (P < 0.05). The expression rate of the Keap1 protein in osteosarcoma tissue was 13.7%, which was significantly lower than the rate in normal peritumour tissue (80.4%). In addition, Nrf2/Keap1 expression was unrelated to patient gender and age, tumour site, and histological type and was related to metastasis and patient response to chemotherapy (P < 0.05). The five-year survival rate was significantly lower in patients with positive Nrf2 expression than in those with negative Nrf2 expression (p = 0.023), and it was significantly higher in patients with positive Keap1 expression than in those with negative Keap1 expression (P = 0.018). Conclusion: The expression of Nrf2-Keap1 is abnormal in osteosarcoma tissue and shows significant clinical relevance for determining the prognosis of osteosarcoma.« less

  15. Prognostic significance of huntingtin interacting protein 1 expression on patients with acute myeloid leukemia

    PubMed Central

    Wang, Jinghan; Yu, Mengxia; Guo, Qi; Ma, Qiuling; Hu, Chao; Ma, Zhixin; Yin, Xiufeng; Li, Xia; Wang, Yungui; Pan, Hanzhang; Wang, Dongmei; Huang, Jiansong; Meng, Haitao; Tong, Hongyan; Qian, Wenbin; Jin, Jie

    2017-01-01

    Huntingtin interacting protein 1 (HIP1) is an endocytic protein which is overexpressed in a variety of human cancers and involved in cancer-causing translocation in leukemia. However, the prognostic impact of HIP1 expression on AML remains unclear. In this study, quantification of HIP1 transcript by real-time quantitative PCR in bone marrow blasts was performed in 270 AML patients. As a result, high HIP1 expression was seen more frequently in older patients, M4/M5 morphology and genes of NPM1 and DNMT3A mutations, and underrepresented in favorable karyotype subgroups and CEBPA double allele mutations in our AML patients. We also found high HIP1 expressers showed lower levels of hemoglobin. In addition, overexpression of HIP1 was associated with an inferior overall survival. The prognostic value of HIP1 expression was validated in patients from an independent TCGA cohort. Notably, up-regulation of miR-16, miR-15a, miR-28 and miR-660 were seen in high HIP1 expressers from the two independent cohorts. In vitro, interfereing of HIP1 expression by siRNA suppressed the proliferation of leukemic cells, and downregulation of these miRNAs were seen in THP-1 and Kasumi cell lines after silencing HIP1 expression. In conclusion, the HIP1 gene expression might serve as a reliable predictor for overall survival in AML patients. PMID:28452374

  16. Prognostic significance of huntingtin interacting protein 1 expression on patients with acute myeloid leukemia.

    PubMed

    Wang, Jinghan; Yu, Mengxia; Guo, Qi; Ma, Qiuling; Hu, Chao; Ma, Zhixin; Yin, Xiufeng; Li, Xia; Wang, Yungui; Pan, Hanzhang; Wang, Dongmei; Huang, Jiansong; Meng, Haitao; Tong, Hongyan; Qian, Wenbin; Jin, Jie

    2017-04-28

    Huntingtin interacting protein 1 (HIP1) is an endocytic protein which is overexpressed in a variety of human cancers and involved in cancer-causing translocation in leukemia. However, the prognostic impact of HIP1 expression on AML remains unclear. In this study, quantification of HIP1 transcript by real-time quantitative PCR in bone marrow blasts was performed in 270 AML patients. As a result, high HIP1 expression was seen more frequently in older patients, M4/M5 morphology and genes of NPM1 and DNMT3A mutations, and underrepresented in favorable karyotype subgroups and CEBPA double allele mutations in our AML patients. We also found high HIP1 expressers showed lower levels of hemoglobin. In addition, overexpression of HIP1 was associated with an inferior overall survival. The prognostic value of HIP1 expression was validated in patients from an independent TCGA cohort. Notably, up-regulation of miR-16, miR-15a, miR-28 and miR-660 were seen in high HIP1 expressers from the two independent cohorts. In vitro, interfereing of HIP1 expression by siRNA suppressed the proliferation of leukemic cells, and downregulation of these miRNAs were seen in THP-1 and Kasumi cell lines after silencing HIP1 expression. In conclusion, the HIP1 gene expression might serve as a reliable predictor for overall survival in AML patients.

  17. [Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

    PubMed

    Yan, Jie; Zhao, Shou-feng; Mao, Ya-fei; Ruan, Ping; Luo, Yi-hui; Li, Shu-ping; Li, Li-wei

    2005-01-01

    To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific

  18. Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

    PubMed Central

    Bandara, G; Mueller, G M; Galea-Lauri, J; Tindal, M H; Georgescu, H I; Suchanek, M K; Hung, G L; Glorioso, J C; Robbins, P D; Evans, C H

    1993-01-01

    Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy. Images Fig. 1 Fig. 2 PMID:8248169

  19. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

    NASA Technical Reports Server (NTRS)

    Gomes, M. D.; Lecker, S. H.; Jagoe, R. T.; Navon, A.; Goldberg, A. L.

    2001-01-01

    Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

  20. Increased expression of stress inducible protein 1 in glioma-associated microglia/macrophages.

    PubMed

    Carvalho da Fonseca, Anna Carolina; Wang, Huaqing; Fan, Haitao; Chen, Xuebo; Zhang, Ian; Zhang, Leying; Lima, Flavia Regina Souza; Badie, Behnam

    2014-09-15

    Factors released by glioma-associated microglia/macrophages (GAMs) play an important role in the growth and infiltration of tumors. We have previously demonstrated that the co-chaperone stress-inducible protein 1 (STI1) secreted by microglia promotes proliferation and migration of human glioblastoma (GBM) cell lines in vitro. In the present study, in order to investigate the role of STI1 in a physiological context, we used a glioma model to evaluate STI1 expression in vivo. Here, we demonstrate that STI1 expression in both the tumor and in the infiltrating GAMs and lymphocytes significantly increased with tumor progression. Interestingly, high expression of STI1 was observed in macrophages and lymphocytes that infiltrated brain tumors, whereas STI1 expression in the circulating blood monocytes and lymphocytes remained unchanged. Our results correlate, for the first time, the expression of STI1 and glioma progression, and suggest that STI1 expression in GAMs and infiltrating lymphocytes is modulated by the brain tumor microenvironment. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity.

    PubMed

    Saupe, Madeleine; Rauschenberger, Lisa; Preuß, Melanie; Oswald, Stefan; Fussek, Sebastian; Zimmermann, Uwe; Walther, Reinhard; Knabbe, Cornelius; Burchardt, Martin; Stope, Matthias B

    2015-10-01

    The development of a drug-resistant phenotype is the major challenge during treatment of castration-resistant prostate cancer (PC). In solid cancer entities, one of the major contributors to chemoresistance is the multidrug resistance 1 (MDR1) protein. Believed to be involved in the induction of MDR1 expression is the presence of anticancer drugs as well as the Y box binding protein 1 (YB-1). Basal as well as drug-induced expression of MDR1 in established PC cell lines was assessed by Western blotting and mass spectrometry. Subsequently, the influence of YB-1 on MDR1 expression was examined via transient overexpression of YB-1. While LNCaP and PC-3 cells showed no detectable amounts of MDR1, the resistance factor was found to be expressed in 22Rv1 cells. Despite this difference, all three cell lines demonstrated similar growth behavior in the presence of the first-line chemotherapeutic agent docetaxel. Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and abiraterone did not significantly alter MDR1 expression levels. Furthermore, overexpression of the MDR1 controlling factor YB-1 showed no impact on MDR1 expression levels. MDR1 was detectable in the PC cell line 22Rv1. However, this study suggests that MDR1 is of less importance for drug resistance in PC cells than in other types of solid cancer. Furthermore, in contrast to YB-1 properties in other malignancies, MDR1 regulation through YB-1 seems to be unlikely.

  2. Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression.

    PubMed

    Franciosa, G; Diluvio, G; Gaudio, F Del; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G Del; Screpanti, I; Checquolo, S

    2016-09-08

    Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4(+)CD8(+) DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL.

  3. Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

    PubMed Central

    Franciosa, G; Diluvio, G; Gaudio, F Del; Giuli, M V; Palermo, R; Grazioli, P; Campese, A F; Talora, C; Bellavia, D; D'Amati, G; Besharat, Z M; Nicoletti, C; Siebel, C W; Choy, L; Rustighi, A; Sal, G Del; Screpanti, I; Checquolo, S

    2016-01-01

    Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL. PMID:26876201

  4. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein*

    PubMed Central

    Chen, Qing; Lu, Mingjian; Monks, Bobby R.; Birnbaum, Morris J.

    2016-01-01

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression. PMID:26668316

  5. Screening and formulation of chemoattractant coatings for artificial reef structures.

    PubMed

    Lee, Han Seong; Sidharthan, M; Shim, Cheol Soo; Kim, Young Do; Lim, Chi Young; Ko, J W; Han, Man Deuk; Rang, Maeng Joo; Bim, Lee Sae; Cho, Hwan Sung; Shin, H W

    2008-07-01

    This study was carried out to augment the colonization of marine benthic communities on artificial reef structure. Increasing marine pollution along with various natural hazards cause severe damages to marine algae and associated fauna. In recent years, artificial reefs have been deployed in coastal regions of several parts of the world in order to increase the marine productivity. They are mainly built with concrete materials, however their leachates have considerable impacts on algae. Therefore to increase the algal colonization five chemoattractants such as ferrous sulfate, zinc oxide, ammonium nitrate, sodium phosphate and ferrous lactate were screened against spores of a fouling alga, Ulva pertusa. FeSO4 / ZnO (8:2) and ferrous lactate coatings showed the highest spore attachment with 52 +/- 5.2 cm2 and 79.5 +/- 10.2 cm2 spores respectively (p<0.01). Furthermore using these chemoattractants, coating formulations were made and their performances were investigated at East coast (Ayajin harbor) and South coast (Meejo harbor) of Korea. A maximum fouling coverage (with green algae 25%, red algae 11.3% and brown algae 63.7%) was estimated from ferrous lactate coatings (p<0.01). Different composition of coating formulations and their chemoattractive properties were evaluated.

  6. CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium.

    PubMed

    Silberg, D G; Furth, E E; Taylor, J K; Schuck, T; Chiou, T; Traber, P G

    1997-08-01

    CDX1 is an intestine-specific transcription factor expressed early in intestinal development that may be involved in regulation of proliferation and differentiation of intestinal epithelial cells. We examined the pattern of CDX1 protein expression in metaplastic and neoplastic tissue to provide insight into its possible role in abnormal differentiation. Tissue samples were stained by immunohistochemistry using an affinity-purified, polyclonal antibody against a peptide epitope of CDX1. Specific nuclear staining was found in epithelial cells of the small intestine and colon. Esophagus and stomach did not express CDX1 protein; however, adjacent areas of intestinal metaplastic tissue intensely stained for CDX1. Adenocarcinomas of the stomach and esophagus had both positive and negative nuclear staining for CDX1. Colonic epithelial cells in adenomatous polyps and adenocarcinomas had a decreased intensity of staining compared with normal colonic crypts in the same specimen. CDX1 may be important in the transition from normal gastric and esophageal epithelium to intestinal-type metaplasia. The variability in expression of CDX1 in gastric and esophageal adenocarcinomas suggests more than one pathway in the development of these carcinomas. The decrease of CDX1 in colonic adenocarcinomas may indicate a role for CDX1 in growth regulation and in the maintenance of the differentiated phenotype.

  7. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma.

    PubMed

    Marsh, Erica E; Chibber, Shani; Wu, Ju; Siegersma, Kendra; Kim, Julie; Bulun, Serdar

    2016-04-01

    To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. RASSF1A protein expression and correlation with clinicopathological parameters in renal cell carcinoma

    PubMed Central

    Tezval, Hossein; Merseburger, Axel S; Matuschek, Ira; Machtens, Stefan; Kuczyk, Markus A; Serth, Jürgen

    2008-01-01

    Background Epigenetic silencing of RAS association family 1A (RASSF1A) tumor suppressor gene occurs in various histological subtypes of renal cell carcinoma (RCC) but RASSF1A protein expression in clear cell RCC as well as a possible correlation with clinicopathological parameters of patients has not been analyzed at yet. Methods 318 primary clear cell carcinomas were analyzed using tissue microarray analysis and immunohistochemistry. Survival analysis was carried out for 187 patients considering a follow-up period of 2–240 month. Results Expression of RASSF1A was found to be significantly decreased in tumoral cells when compared to normal tubular epithelial cells. RASSF1A immunopositivity was significantly associated with pT stage, group stage and histological grade of tumors and showed a tendency for impaired survival in Kaplan-Meier analysis. Conclusion While most tumors demonstrate a loss of RASSF1A protein, a subset of tumors was identified to exhibit substantial RASSF1A protein expression and show increased tumor progression. Thus RCC tumorigenesis without depletion of RASSF1A may be associated with an adverse clinical outcome. PMID:18822131

  9. Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

    PubMed

    Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

    2017-08-31

    C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

  10. Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

    PubMed Central

    Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

    2017-01-01

    C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

  11. N-Caffeoyltryptamine, a Potent Anti-Inflammatory Phenolic Amide, Suppressed MCP-1 Expression in LPS-stimulated THP-1 Cells and Rats Fed a High-Fat Diet.

    PubMed

    Park, Jae B

    2017-05-27

    Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of several inflammatory diseases including arthrosclerosis. N -caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential effect of N -caffeoyltryptamine on MCP-1 expression was investigated as a potential p38 mitogen-activated protein (MAP) kinase inhibitor in vitro and in vivo. At the concentration of 20 μM, N -caffeoyltryptamine significantly inhibited p38 MAP kinase α, β, γ and δ by 15-50% ( p < 0.05), particularly p38 MAP kinase α (IC 50 = 16.7 μM) and β (IC 50 = 18.3 μM). Also, the pretreatment of the lipopolysaccharide (LPS)-stimulated THP-1 cells with N -caffeoyltryptamine (10, 20 and 40 μM) led to significant suppression of MCP-1 production by 10-45% ( p < 0.05) in the cells. Additionally, N -caffeoyltryptamine was also able to significantly downregulate MCP-1 mRNA expression in the THP-1 cells ( p < 0.05). On the basis of this strong inhibition in vitro, an animal study was conducted to confirm this inhibitory effect in vivo. Rats were divided into three groups ( n = 8): a normal control diet (C), a high-fat diet (HF), or a high-fat diet supplemented with N -caffeoyltryptamine (2 mg per day) (HFS). After 16 weeks, blood samples were collected from the rats in each group, and MCP-1 levels were determined in plasma with other atherogenic markers (C-reactive protein and soluble E-selectin (sE-selectin)). As expected, the average MCP-1 levels of the HF group were found to be higher than those of the C group ( p < 0.05). However, the MCP-1 levels of the HFS group were significantly lower than those of the HF group ( p < 0.05), suggesting that N -caffeoyltryptamine could decrease MCP-1 expression in vivo. Related to other atherogenic markers such as C-reactive protein and sE-selectin, there was no significant difference in their levels between the HF and HFS groups

  12. Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

    PubMed

    Gao, Bihu; Kikuchi-Utsumi, Kazue; Ohinata, Hiroshi; Hashimoto, Masaaki; Kuroshima, Akihiro

    2003-06-01

    Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

  13. Prognostic value of decreased FOXP1 protein expression in various tumors: a systematic review and meta-analysis

    PubMed Central

    Xiao, Jian; He, Bixiu; Zou, Yong; Chen, Xi; Lu, Xiaoxiao; Xie, Mingxuan; Li, Wei; He, Shuya; You, Shaojin; Chen, Qiong

    2016-01-01

    The prognostic value of forkhead box protein P1 (FOXP1) protein expression in tumors remains controversial. Therefore, we conducted a systematic review and meta-analysis, searching the PubMed, Embase and Web of Science databases to identify eligible studies. In total, we analyzed 22 articles that examined 9 tumor types and included 2468 patients. Overall, decreased expression of FOXP1 protein was associated with favorable overall survival (OS) in lymphoma patients (HR = 0.38, 95%CI: 0.30–0.48, p < 0.001). In patients with solid tumors, decreased FOXP1 expression correlated with unfavorable OS (HR = 1.82, 95%CI: 1.18–2.83, p = 0.007). However, when FOXP1 protein expression was nuclear, decreased expression was also associated with favorable OS (HR = 0.53, 95%CI: 0.32–0.86, p = 0.011). Furthermore, decreased FOXP1 expression resulted in the best OS in patients with mucosa-associated lymphoid tissue (MALT) lymphomas (HR = 0.26, 95%CI: 0.11–0.59, p = 0.001), but the worst OS was observed in non-small cell lung cancer (NSCLC) patients (HR = 3.11, 95%CI: 1.87–5.17, p < 0.001). In addition, decreased FOXP1 expression was significantly correlated with an unfavorable relapse-free survival (RFS) in breast cancer patients (HR = 1.93, 95%CI: 1.33–2.80, p = 0.001). PMID:27457567

  14. Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

    PubMed Central

    Warenius, H M; Jones, M; Gorman, T; McLeish, R; Seabra, L; Barraclough, R; Rudland, P

    2000-01-01

    The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign PMID:10993658

  15. Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice

    PubMed Central

    2013-01-01

    Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. Results To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP. Conclusions We have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1. PMID:23971992

  16. Prenatal low-dose methylmercury exposure impairs neurite outgrowth and synaptic protein expression and suppresses TrkA pathway activity and eEF1A1 expression in the rat cerebellum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako; Cheng, Jinping

    Methylmercury (MeHg) is a highly neurotoxic environmental chemical that can cause developmental impairments. Human fetuses and neonates are particularly susceptible to MeHg toxicity; however, the mechanisms governing its effects in the developing brain are unclear. In the present study, we investigated the effects of prenatal and lactational MeHg exposure on the developing cerebellum in rats. We demonstrated that exposure to 5 ppm MeHg decreased postnatal expression of pre- and postsynaptic proteins, suggesting an impairment in synaptic development. MeHg exposure also reduced neurite outgrowth, as shown by a decrease in the expression of the neurite marker neurofilament H. These changes weremore » not observed in rats exposed to 1 ppm MeHg. In order to define the underlying mechanism, we investigated the effects of MeHg exposure on the tropomyosin receptor kinase (Trk) A pathway, which plays important roles in neuronal differentiation and synapse formation. We demonstrated suppression of the TrkA pathway on gestation day 20 in rats exposed to 5 ppm MeHg. In addition, down-regulation of eukaryotic elongation factor 1A1 (eEF1A1) was observed on postnatal day 1. eEF1A1 knockdown in differentiating PC12 cells impaired neurite outgrowth and synaptic protein expression, similar to the results of MeHg exposure in the cerebellum. These results suggest that suppression of the TrkA pathway and subsequent decreases in eEF1A1 expression induced by prenatal exposure to MeHg may lead to reduced neurite outgrowth and synaptic protein expression in the developing cerebellum. - Highlights: • Prenatal exposure to MeHg decreased postnatal expression of synaptic proteins. • MeHg exposure also reduced neurite outgrowth postnatally. • Suppression of the TrkA pathway and eEF1A1 expression was induced by MeHg exposure. • eEF1A1 knockdown impaired neurite outgrowth and synaptic protein expression.« less

  17. Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter.

    PubMed

    Biamonti, G; Bassi, M T; Cartegni, L; Mechta, F; Buvoli, M; Cobianchi, F; Riva, S

    1993-03-05

    hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

  18. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

    PubMed

    Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

    2000-03-01

    Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

  19. Expression of nuclear receptor interacting proteins TIF-1, SUG-1, receptor interacting protein 140, and corepressor SMRT in tamoxifen-resistant breast cancer.

    PubMed

    Chan, C M; Lykkesfeldt, A E; Parker, M G; Dowsett, M

    1999-11-01

    Regulation of gene transcription as a consequence of steroid receptor-DNA interaction is mediated via nuclear receptor interacting proteins (RIPs), including coactivator or corepressor proteins, which interact with both the receptor and components of the basic transcriptional unit and vary between cell types. The aim of this study was to test the hypothesis that resistance of some breast carcinomas to tamoxifen was associated with inappropriate expression of some of these RIPs. Using Northern analysis, we observed no significant difference between the amount of either TIF-1 or SUG-1 mRNA expressed in parental MCF-7 and MCF-7 tamoxifen-resistant cell lines. However, the expression of RIP140 mRNA was lower in the resistant cell line and in the presence of estradiol, the level of RIP140 mRNA was higher in the resistant cells but not in the parental cells. In a cohort of 19 tamoxifen-resistant breast tumor samples, there was no significant difference in the level of the RIP140 and TIF-1 and corepressor SMRT mRNA compared with tamoxifen-treated tumors (n = 6) or untreated tumors (n = 21). However, SUG-1 mRNA was lower in resistant breast tumors. These data provide no support for increased expression of these RIPs or decreased expression of corepressor SMRT for being a mechanism for resistance of breast tumors to tamoxifen.

  20. Heat sensitization in skin and muscle nociceptors expressing distinct combinations of TRPV1 and TRPV2 protein.

    PubMed

    Rau, K K; Jiang, N; Johnson, R D; Cooper, B Y

    2007-04-01

    Recordings were made from small and medium diameter dorsal root ganglia (DRG) neurons that expressed transient receptor potential (TRP) proteins. Physiologically characterized skin nociceptors expressed either TRPV1 (type 2) or TRPV2 (type 4) in isolation. Other nociceptors co-expressed both TRP proteins and innervated deep tissue sites (gastrocnemius muscle, distal colon; type 5, type 8) and skin (type 8). Subpopulations of myelinated (type 8) and unmyelinated (type 5) nociceptors co-expressed both TRPs. Cells that expressed TRPV1 were excellent transducers of intense heat. Proportional inward currents were obtained from a threshold of approximately 46.5 to approximately 56 degrees C. In contrast, cells expressing TRPV2 alone (52 degrees C threshold) did not reliably transduce the intensity of thermal events. Studies were undertaken to assess the capacity of skin and deep nociceptors to exhibit sensitization to repeated intense thermal stimuli [heat-heat sensitization (HHS)]. Only nociceptors that expressed TRPV2, alone or in combination with TRPV1, exhibited HHS. HHS was shown to be Ca(2+) dependent in either case. Intracellular Ca(2+) dependent pathways to HHS varied with the pattern of TRP protein expression. Cells co-expressing both TRPs modulated heat reactivity through serine/threonine phosphorylation or PLA(2)-dependent pathways. Cells expressing only TRPV2 may have relied on tyrosine kinases for HHS. We conclude that heat sensitization in deep and superficial capsaicin and capsaicin-insensitive C and Adelta nociceptors varies with the distribution of TRPV1 and TRPV2 proteins. The expression pattern of these proteins are specific to subclasses of physiologically identified C and A fiber nociceptors with highly restricted tissue targets.

  1. Fumaric acid attenuates the eotaxin-1 expression in TNF-α-stimulated fibroblasts by suppressing p38 MAPK-dependent NF-κB signaling.

    PubMed

    Roh, Kyung-Baeg; Jung, Eunsun; Park, Deokhoon; Lee, Jongsung

    2013-08-01

    Eotaxin-1 is a potent chemoattractant for eosinophils and a critical mediator during the development of eosinophilic inflammation. Fumaric acid is an intermediate product of the citric acid cycle, which is source of intracellular energy. Although fumaric acid ameliorates psoriasis and multiple sclerosis, its involvement in eotaxin-1-mediated effects has not been assessed. In this study, we investigated the effects of fumaric acid on eotaxin-1 expression in a mouse fibroblast cell line. We found that fumaric acid significantly inhibited tumor necrosis factor-α (TNF-α-induced eotaxin-1 expression. This fumaric acid effect was mediated through the inhibition of p38 mitogen-activated protein kinase (MAPK)-dependent nuclear factor (NF)-κB signaling. We also found that fumaric acid operates downstream of MEKK3 during TNF-α-induced NF-κB signaling, which upregulated eotaxin-1 expression. In addition, fumaric acid attenuated expression of CC-chemokine receptor 3 (CCR3), an eotaxin-1 receptor, and adhesion molecules that play important roles in eosinophil binding to induce allergic inflammation. Taken together, these findings indicate that inhibiting TNF-α-induced eotaxin-1 expression by fumaric acid occurs primarily through suppression of NF-κB signaling, which is mediated by inhibiting p38 MAPK and suggest that fumaric acid may be used as a complementary treatment option for eotaxin-1-mediated diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Osthole Attenuates Inflammatory Responses and Regulates the Expression of Inflammatory Mediators in HepG2 Cells Grown in Differentiated Medium from 3T3-L1 Preadipocytes.

    PubMed

    Wu, Shu-Ju

    2015-09-01

    This study explored the anti-inflammatory mechanisms by which osthole acted on HepG2 cells cultured in a differentiated medium from cultured 3T3-L1 preadipocyte cells. HepG2 cells, a human liver cell line, were treated with various concentrations of osthole in differentiated media from cultured 3T3-L1 cells to evaluate proinflammatory cytokines, inflammatory mediators, and signaling pathways. We used enzyme-linked immunosorbent assay kits to determine the levels of proinflammatory cytokines, real-time polymerase chain reaction to assay the mRNA expression, and western blot to determine the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) proteins. We also investigated inflammatory mechanism pathway members, including mitogen-activated protein kinase (MAPK) and nuclear transcription factor kappa-B (NF-κB). Osthole was able to suppress the levels of proinflammatory cytokines interleukin (IL)-1β and IL-6, as well as chemokines monocyte chemoattractant protein-1 and IL-8. In addition, COX-2 was suppressed and HO-1 expression was increased in a concentration-dependent manner. Osthole was also able to decrease IκB-α phosphorylation and suppress the phosphorylation of MAPKs. These results suggest that osthole has anti-inflammatory effects as demonstrated by the decreased proinflammatory cytokine and mediator production through suppression of the NF-κB and MAPK signaling pathways in HepG2 cells when they are incubated on the differentiated medium from 3T3-L1 cells.

  3. Selenium-binding protein 1: a sensitive urinary biomarker to detect heavy metal-induced nephrotoxicity.

    PubMed

    Lee, Eui Kyung; Shin, Young-Jun; Park, Eun Young; Kim, Nam Deuk; Moon, Aree; Kwack, Seung Jun; Son, Ji Yeon; Kacew, Sam; Lee, Byung Mu; Bae, Ok-Nam; Kim, Hyung Sik

    2017-04-01

    Identifying novel biomarkers to detect nephrotoxicity is clinically important. Here, we attempted to identify new biomarkers for mercury-induced nephrotoxicity and compared their sensitivity to that of traditional biomarkers in animal models. Comparative proteomics analysis was performed in kidney tissues of Sprague-Dawley rats after oral treatment with HgCl 2 (0.1, 1, or 5 mg/kg/day) for 21 days. Kidney cortex tissues were analyzed by two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization, and differentially expressed proteins were identified. The corresponding spots were quantitated by RT-PCR. Selenium-binding protein 1 (SBP1) was found to be the most markedly upregulated protein in the kidney cortex of rats after HgCl 2 administration. However, blood urea nitrogen, serum creatinine, and glucose levels increased significantly only in the 1 or 5 mg/kg HgCl 2 -treated groups. A number of urinary excretion proteins, including kidney injury molecule-1, clusterin, monocyte chemoattractant protein-1, and β-microglobulin, increased dose-dependently. Histopathological examination revealed severe proximal tubular damage in high-dose (5 mg/kg) HgCl 2 -exposed groups. In addition, urinary excretion of SBP1 significantly increased in a dose-dependent manner. To confirm the critical role of SBP1 as a biomarker for nephrotoxicity, normal kidney proximal tubular cells were treated with HgCl 2 , CdCl 2 , or cisplatin for 24 h. SBP1 levels significantly increased in conditioned media exposed to nephrotoxicants, but decreased in cell lysates. Our investigations suggest that SBP1 may play a critical role in the pathological processes underlying chemical-induced nephrotoxicity. Thus, urinary excretion of SBP1 might be a sensitive and specific biomarker to detect early stages of kidney injury.

  4. Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71.

    PubMed

    Chen, Hsuan-Fu; Chang, Meng-Huei; Chiang, Bor-Luen; Jeng, Shih-Tong

    2006-04-05

    Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease associated with fatal neurological complications in young children, and several major outbreaks have occurred recently. This study developed an effective antiviral agent by transforming the gene for VP1 protein, a previously defined epitope and also a coat protein of EV71, into tomato plant. VP1 protein was first fused with sorting signals to enable it to be retained in the endoplasmic reticulum of tomato plant, and its expression level increased to 27 microg/g of fresh tomato fruit. Transgenic tomato fruit expressing VP1 protein was then used as an oral vaccine, and the development of VP1-specific fecal IgA and serum IgG were observed in BALB/c mice. Additionally, serum from mice fed transgenic tomato could neutralize the infection of EV71 to rhabdomyosarcoma cells, indicating that tomato fruit expressing VP1 was successful in orally immunizing mice. Moreover, the proliferation of spleen cells from orally immunized mice was stimulated by VP1 protein, and provided further evidence of both humoral and cellular immunity. Results of this study not only demonstrate the feasibility of using transgenic tomato as an oral vaccine to generate protective immunity in mice against EV71, but also suggest the probability of enterovirus vaccine development.

  5. Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

    PubMed Central

    Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

    2000-01-01

    The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660

  6. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    PubMed

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  7. Transcription factor specificity protein 1 modulates TGFβ1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

    PubMed

    Yamaguchi, Rui; Sakamoto, Arisa; Yamaguchi, Reona; Haraguchi, Misa; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2018-08-01

    The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor β1 (TGFβ1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFβ1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFβ1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFβ1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFβ1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFβ1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFβ1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPβ or TIF1β siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFβ1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFβ1/Smad signaling and

  8. Expression of autophagy-related protein beclin-1 in malignant canine mammary tumors

    PubMed Central

    2013-01-01

    Background Autophagy is a self-catabolic mechanism that degrades unnecessary cellular components through lysosomal enzymes. Beclin-1, an autophagy-related protein, establishes the first connection between autophagy and tumorigenesis. The purpose of this study is to assess the Beclin-1 expression pattern and to determine its prognostic significance in patients with malignant canine mammary tumor (CMT). Results We examined Beclin-1 expression in 70 cases of malignant CMTs by immunohistochemistry. Cytoplasmic Beclin-1 expression was significantly weaker in cancer cells than in nearby normal mammary glands (p < 0.001). Low cytoplasmic expression (57.14%) was associated with older age, lower degree of tubular formation, increased mitotic activity, higher histologic grade, and extensive necrosis. Low nuclear expression (40%) was connected with older age, lower degree of tubular formation, extensive necrosis, and negative for Her2/neu overexpression. Univariate survival analysis showed that Beclin-1 cytoplasmic expression was a poor prognostic factor for overall survival rate (p < 0.001). Multivariate survival analysis demonstrated that Beclin-1 cytoplasmic expression is an independent prognostic factor (p = 0.016). Conclusions Loss of Beclin-1 is associated with aggressive clinicopathologic features and poor overall survival. The results suggest that Beclin-1 plays an important role in tumor progression of malignant CMTs. PMID:23578251

  9. Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

    PubMed

    Kimura, Yukihiro; Chihara, Kazuyasu; Honjoh, Chisato; Takeuchi, Kenji; Yamauchi, Shota; Yoshiki, Hatsumi; Fujieda, Shigeharu; Sada, Kiyonao

    2014-11-07

    Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Regulation of the expression of plant resistance gene SNC1 by a protein with a conserved BAT2 domain.

    PubMed

    Li, Yingzhong; Tessaro, Mark J; Li, Xin; Zhang, Yuelin

    2010-07-01

    Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level.

  11. Direct inhibition of RNAse T2 expression by the HTLV-1 viral protein Tax.

    PubMed

    Polakowski, Nicholas; Han, Hongjin; Lemasson, Isabelle

    2011-08-01

    Adult T-cell leukemia (ATL) is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1) infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP) assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  12. Direct Inhibition of RNAse T2 Expression by the HTLV-1 Viral Protein Tax

    PubMed Central

    Polakowski, Nicholas; Han, Hongjin; Lemasson, Isabelle

    2011-01-01

    Adult T-cell leukemia (ATL) is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1) infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP) assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development. PMID:21994792

  13. Tax Protein-induced Expression of Antiapoptotic Bfl-1 Protein Contributes to Survival of Human T-cell Leukemia Virus Type 1 (HTLV-1)-infected T-cells*♦

    PubMed Central

    Macaire, Héloïse; Riquet, Aurélien; Moncollin, Vincent; Biémont-Trescol, Marie-Claude; Duc Dodon, Madeleine; Hermine, Olivier; Debaud, Anne-Laure; Mahieux, Renaud; Mesnard, Jean-Michel; Pierre, Marlène; Gazzolo, Louis; Bonnefoy, Nathalie; Valentin, Hélène

    2012-01-01

    Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4+ T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-xL, and Bcl-2. Indeed, both Bfl-1 and Bcl-xL knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-xL in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-xL represent potential therapeutic targets for ATLL treatment. PMID:22553204

  14. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

    PubMed

    Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

    2016-01-01

    Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

  15. Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells.

    PubMed

    Ryter, Stefan W; Xi, Sichuan; Hartsfield, Cynthia L; Choi, Augustine M K

    2002-08-01

    Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.

  16. Nitric oxide increases Wnt-induced secreted protein-1 (WISP-1/CCN4) expression and function in colitis.

    PubMed

    Wang, Hongying; Zhang, Rui; Wen, Shoubin; McCafferty, Donna-Marie; Beck, Paul L; MacNaughton, Wallace K

    2009-04-01

    Nitric oxide (NO) derived from the inducible NO synthase (iNOS) is an important and complex mediator of inflammation in the intestine. Wnt-inducible secreted protein (WISP)-1 (CCN4), a member of the connective tissue growth factor family, is involved in tissue repair. We sought to determine the relationship between iNOS and WISP-1 in colitis. By analyzing human colonic biopsy samples, we showed that the expression of mRNA for both iNOS and WISP-1 was significantly higher in ulcerative colitis samples compared with control tissue. The upregulation of WISP-1 was positively correlated with iNOS expression in two models of colitis, induced by intrarectal trinitrobenzenesulfonic acid (TNBS) or occurring spontaneously in IL-10 deficient mice. Loss of iNOS, studied using iNOS(-/-) mice in both TNBS-induced and IL-10(-/-) colitis models, significantly attenuated the colitis-related WISP-1 increase. In human colonic epithelial cell lines, the NO donor, DETA-NONOate, elevated WISP-1 mRNA and protein expression through a beta-catenin and CREB-dependent, but Wnt-1-independent, pathway. In addition, NO-induced WISP-1 directly induced secretion of soluble collagen in colonic fibroblast cells. NO increases WISP-1 expression both in vitro and in vivo, suggesting a new role for iNOS and NO in colitis.

  17. WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway.

    PubMed

    Zhou, Bo; Wang, Dexuan; Feng, Xiuyan; Zhang, Yiqian; Wang, Yanhui; Zhuang, Jieqiu; Zhang, Xuemei; Chen, Guangping; Delpire, Eric; Gu, Dingying; Cai, Hui

    2012-03-01

    WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.

  18. Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

    PubMed Central

    Lee, Jinhwa; Yu, Hai; Li, Yonghai; Ma, Jingjiao; Lang, Yuekun; Duff, Michael; Henningson, Jamie; Liu, Qinfang; Li, Yuhao; Nagy, Abdou; Bawa, Bhupinder; Li, Zejun; Tong, Guangzhi; Richt, Juergen A.; Ma, Wenjun

    2017-01-01

    Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses. PMID:28142079

  19. Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

    PubMed

    Rehan, Shahid; Jaakola, Veli-Pekka

    2015-10-01

    Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

    PubMed

    Petrie Aronin, Caren E; Zhao, Yun M; Yoon, Justine S; Morgan, Nicole Y; Prüstel, Thorsten; Germain, Ronald N; Meier-Schellersheim, Martin

    2017-11-21

    Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments. Published by Elsevier Inc.

  1. MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

    PubMed

    Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

    2002-03-20

    We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

  2. The Effect of Post-Resistance Exercise Amino Acids on Plasma MCP-1 and CCR2 Expression

    PubMed Central

    Wells, Adam J.; Hoffman, Jay R.; Jajtner, Adam R.; Varanoske, Alyssa N.; Church, David D.; Gonzalez, Adam M.; Townsend, Jeremy R.; Boone, Carleigh H.; Baker, Kayla M.; Beyer, Kyle S.; Mangine, Gerald T.; Oliveira, Leonardo P.; Fukuda, David H.; Stout, Jeffrey R.

    2016-01-01

    The recruitment and infiltration of classical monocytes into damaged muscle is critical for optimal tissue remodeling. This study examined the effects of an amino acid supplement on classical monocyte recruitment following an acute bout of lower body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm) ingested supplement (SUPP) or placebo (PL) immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL), immediately (IP), 30-min (30P), 1-h (1H), 2-h (2H), and 5-h (5H) post-exercise to assess plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), myoglobin, cortisol and insulin concentrations; and expressions of C-C chemokine receptor-2 (CCR2), and macrophage-1 antigen (CD11b) on classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Changes in myoglobin, cortisol, and insulin concentrations were similar between treatments. Compared to PL, plasma MCP-1 was “very likely greater” (98.1% likelihood effect) in SUPP at 2H. CCR2 expression was “likely greater” at IP (84.9% likelihood effect), “likely greater” at 1H (87.7% likelihood effect), “very likely greater” at 2H (97.0% likelihood effect), and “likely greater” at 5H (90.1% likelihood effect) in SUPP, compared to PL. Ingestion of SUPP did not influence CD11b expression. Ingestion of an amino acid supplement immediately post-exercise appears to help maintain plasma MCP-1 concentrations and augment CCR2 expression in resistance trained men. PMID:27384580

  3. A novel apoptosis correlated molecule: expression and characterization of protein Latcripin-1 from Lentinula edodes C(91-3).

    PubMed

    Liu, Ben; Zhong, Mintao; Lun, Yongzhi; Wang, Xiaoli; Sun, Wenchang; Li, Xingyun; Ning, Anhong; Cao, Jing; Zhang, Wei; Liu, Lei; Huang, Min

    2012-01-01

    An apoptosis correlated molecule-protein Latcripin-1 of Lentinula edodes C(91-3)-was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C(91-3). According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3'-Full Rapid Amplification of cDNA Ends (RACE) and 5'-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.

  4. Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

    PubMed Central

    Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

    2008-01-01

    The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

  5. BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

    PubMed

    Arsov, I; Li, X; Matthews, G; Coradin, J; Hartmann, B; Simon, A K; Sealfon, S C; Yue, Z

    2008-09-01

    Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

  6. Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco.

    PubMed

    Zhang, J-Y; Qiao, Y-S; Lv, D; Gao, Z-H; Qu, S-C; Zhang, Z

    2012-03-01

    Most commercially grown apple cultivars are susceptible to fungal diseases. Malus hupehensis has high resistance to many diseases affecting apple cultivars. Understanding innate defence mechanisms would help to develop disease-resistant apple crops. Non-expressor of pathogenesis-related genes 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). MhNPR1 cDNA, corresponding to genomic DNA and its 5' flanking sequences, was isolated from M. hupehensis. Sequence analysis showed that the regulatory mechanism for oligomer-monomer transition of the MhNPR1 protein in apple might be similar to that of GmNPR1 in soybean, but different from that of AtNPR1 in Arabidopsis. No significant differences in MhNPR1 expression were found in M. hupehensis after infection with Botryosphaeria berengeriana, showing that MhNPR1 might be regulated by pathogens at the protein level, as described for Arabidopsis and grapevine. SA treatment significantly induced MhNPR1 expression in leaves, stems and roots, while methyl jasmonate (MeJA) treatment induced MhNPR1 expression in roots, but not in leaves or stems. The expression of MhNPR1 was highly increased in roots, moderately in leaves, and did not change in stems after treatment with 1-aminocyclopropane-1-carboxylic acid (ACC). SAR marker genes (MhPR1 and MhPR5) were induced by SA, MeJA and ACC in leaves, stems and roots. Overexpression of MhNPR1 significantly induced the expression of pathogenesis-related genes (NtPR1, NtPR3 and NtPR5) in transgenic tobacco plants and resistance to the fungus Botrytis cinerea, suggesting that MhNPR1 orthologues are a component of the SA defence signalling pathway and SAR is induced in M. hupehensis. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

  7. SIRT1 Polymorphisms and Serum-Induced SIRT1 Protein Expression in Aging and Frailty: The CHAMP Study.

    PubMed

    Razi, Shajjia; Cogger, Victoria C; Kennerson, Marina; Benson, Vicky L; McMahon, Aisling C; Blyth, Fiona M; Handelsman, David J; Seibel, Markus J; Hirani, Vasant; Naganathan, Vasikaran; Waite, Louise; de Cabo, Rafael; Cumming, Robert G; Le Couteur, David G

    2017-07-01

    The nutrient sensing protein, SIRT1 influences aging and nutritional interventions such as caloric restriction in animals, however, the role of SIRT1 in human aging remains unclear. Here, the role of SIRT1 single-nucleotide polymorphisms (SNPs) and serum-induced SIRT1 protein expression (a novel assay that detects circulating factors that influence SIRT1 expression in vitro) were studied in the Concord Health and Ageing in Men Project (CHAMP), a prospective cohort of community dwelling men aged 70 years and older. Serum-induced SIRT1 expression was not associated with age or mortality, however participants within the lowest quintile were less likely to be frail (odds ratio (OR) 0.34, 95% confidence interval (CI) 0.17-0.69, N = 1,309). Serum-induced SIRT1 expression was associated with some markers of body composition and nutrition (height, weight, body fat and lean % mass, albumin, and cholesterol) but not disease. SIRT1 SNPs rs2273773, rs3740051, and rs3758391 showed no association with age, frailty, or mortality but were associated with weight, height, body fat and lean, and albumin levels. There were some weak associations between SIRT1 SNPs and arthritis, heart attack, deafness, and cognitive impairment. There was no association between SIRT1 SNPs and the serum-induced SIRT1 assay. SIRT1 SNPs and serum-induced SIRT1 expression in older men may be more closely associated with nutrition and body composition than aging and age-related conditions. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Genes and Proteins Differentially Expressed during In Vitro Malignant Transformation of Bovine Pancreatic Duct Cells1

    PubMed Central

    Jesnowski, R; Zubakov, Dmitri; Faissner, Ralf; Ringel, Jörg; Hoheisel, Jörg D; Lösel, Ralf; Schnölzer, Martina; Löhr, Matthias

    2007-01-01

    Abstract Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDIα), up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation. PMID:17356710

  9. T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

    PubMed Central

    Taub, D D; Anver, M; Oppenheim, J J; Longo, D L; Murphy, W J

    1996-01-01

    IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID:8621778

  10. Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1).

    PubMed

    Seki, N; Muramatsu, M; Sugano, S; Suzuki, Y; Nakagawara, A; Ohhira, M; Hayashi, A; Hori, T; Saito, T

    1998-01-01

    Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

  11. IL-1β mediating high mobility group box protein-1 expression in condylar chondrocyte during temporomandibular joint inflammation.

    PubMed

    Li, Cheng; Cai, Hengxing; Meng, Qinggong; Feng, Yaping; Guo, Huilin; Fang, Wei; Long, Xing

    2016-08-01

    Temporomandibular joint (TMJ) osteoarthritis(OA)characterized with cartilage degen-eration is associated with inflammation. High mobility group box chromosomal protein-1(HMGB-1)is a potent mediator of inflammation and the trigger of OA. The expression of HMGB-1 in TMJ OA was uncovered, but the role of HMGB-1 in TMJ cartilage degeneration is not fully understood. In this study, the regulation of HMGB-1 in TMJ condylar cartilage was revealed. A complete Freund's adjuvant (CFA)-induced TMJ inflammation animal model was employed and the expression of HMGB-1 was detected at 1st, 2nd, and 6th weeks by immunohistochemistry. TMJ condylar chondrocytes were incubated with IL-1β (10 and 40 ng/ml) at 24, 48, and 72 h, and the translocation and protein level of HMGB-1 were evaluated by immunofluorescence and Western blot. Nuclear HMGB-1 staining was predominantly located in chondrocytes of both the fibrosis and proliferative zones in healthy TMJ. 1st week and 2nd week after CFA injection, immunoreaction could be detected in the cytoplasms of HMGB-1-positive cells and cartilage matrix especially in hypertrophic zone. At 6th week after CFA injection, cartilage matrix expression was disappeared and the cytoplasm expression of HMGB-1 was very weak in hypertrophic zone. HMGB-1 was translocated from the nucleus to the cytoplasm at 48 h after incubated with IL-1β (10 ng/ml and 40 ng/ml). The protein level of HMGB-1 was increased after stimulation and had a peak at 48 h. HMGB-1 might be associated with TMJ inflammation and OA. Insight into the role of HMGB-1 in TMJ inflammation is helpful to add the new knowledge into the pathogenesis of TMJ OA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Bone Morphogenetic Protein 3 Controls Insulin Gene Expression and Is Down-regulated in INS-1 Cells Inducibly Expressing a Hepatocyte Nuclear Factor 1A–Maturity-onset Diabetes of the Young Mutation*

    PubMed Central

    Bonner, Caroline; Farrelly, Angela M.; Concannon, Caoimhín G.; Dussmann, Heiko; Baquié, Mathurin; Virard, Isabelle; Wobser, Hella; Kögel, Donat; Wollheim, Claes B.; Rupnik, Marjan; Byrne, Maria M.; König, Hans-Georg; Prehn, Jochen H. M.

    2011-01-01

    Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A–maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY. PMID:21628466

  13. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

    PubMed Central

    Margres, Mark J.; Wray, Kenneth P.; Seavy, Margaret; McGivern, James J.; Herrera, Nathanael D.; Rokyta, Darin R.

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  14. Chlorella 11-Peptide Inhibits the Production of Macrophage-Induced Adhesion Molecules and Reduces Endothelin-1 Expression and Endothelial Permeability

    PubMed Central

    Shih, Mei Fen; Chen, Lih Chi; Cherng, Jong Yuh

    2013-01-01

    The inflammation process in large vessels involves the up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which are also known as the markers of atherosclerosis. We have reported that Chlorella 11-peptide exhibited effective anti-inflammatory effects. This peptide with an amino sequence Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe was further examined for its potential in preventing atherosclerosis in this study. In particular, the roles of Chlorella 11-peptide in lowering the production of vascular adhesion molecules, monocyte chemoattractant protein (MCP-1) and expression of endothelin-1 (ET-1) from endothelia (SVEC4-10 cells) were studied. The production of E-selectin, ICAM-1, VCAM-1 and MCP-1 in SVEC4-10 cells was measured with ELISA. The mRNA expression of ET-1 was analyzed by RT-PCR and agarose gel. Results showed that Chlorella 11-peptide significantly suppressed the levels of E-selectin, ICAM, VCAM, MCP-1 as well as ET-1 gene expression. The inhibition of ICAM-1 and VCAM-1 production by Chlorella 11-peptide was reversed in the presence of protein kinase A inhibitor (H89) which suggests that the cAMP pathway was involved in the inhibitory cause of the peptide. In addition, this peptide was shown to reduce the extent of increased intercellular permeability induced by combination of 50% of lipopolysaccharide (LPS)-activated RAW 264.7 cells medium and 50% normal SEVC cell culture medium (referred to as 50% RAW-conditioned medium). These data demonstrate that Chlorella 11-peptide is a promising biomolecule in preventing chronic inflammatory-related vascular diseases. PMID:24129228

  15. Brassicaceae Express Multiple Isoforms of Biotin Carboxyl Carrier Protein in a Tissue-Specific Manner1

    PubMed Central

    Thelen, Jay J.; Mekhedov, Sergei; Ohlrogge, John B.

    2001-01-01

    Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity. PMID:11299381

  16. Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

    PubMed

    Guo, Hui; Cao, Cuili; Chi, Xueqian; Zhao, Junxia; Liu, Xia; Zhou, Najing; Han, Shuo; Yan, Yongxin; Wang, Yanling; Xu, Yannan; Yan, Yunli; Cui, Huixian; Sun, Hongxia

    2014-10-01

    Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells. © 2014 Wiley Periodicals, Inc.

  17. A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

    PubMed

    Abraham, S; Solomon, W B

    2000-09-19

    We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

  18. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

    PubMed Central

    Nora, Tamara; Bouchonnet, Francine; Labrosse, Béatrice; Charpentier, Charlotte; Mammano, Fabrizio; Clavel, François; Hance, Allan J

    2008-01-01

    Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env) replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1). Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70), and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799), soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5) was also evaluated for a subset of the recombinant viruses (n = 20). Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in characterizing the

  19. [The expression of thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) protein in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy].

    PubMed

    Ishibashi, Keiichiro; Okada, Norimichi; Ishiguro, Toru; Kuwabara, Kouki; Ohsawa, Tomonori; Yokoyama, Masaru; Kumamoto, Kensuke; Haga, Norihiro; Mori, Takashi; Yamada, Hirofumi; Miura, Ichiro; Tamaru, Junichi; Itoyama, Shinji; Ishida, Hideyuki

    2010-11-01

    Thymidylate synthase (TS) and excision repair complementing-1 (ERCC-1) were known to be important biomarkers to predict a tumor response to 5-fluorouracil (5-FU) and oxaliplatin, but the relationship between these expressions and tumor response were still unclear. The aim of this study was to determine whether the expression of TS and ERCC-1 protein predict a tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy as first-line treatment. Fifty patients with unresectable colorectal cancer treated with mFOLFOX6 therapy were enrolled in this study. The expression of TS and ERCC-1 protein in primary cancer cells were examined using immunohistochemistry. There were no significant differences between response rate and the expression of TS or ERCC-1 protein (TS: p>0.99, ERCC-1: p= 0.50). There were no significant differences between progression-free survival time and the expression of TS or ERCC-1 protein (TS: p=0.60, ERCC-1: p=0.60). In this study, the expression TS and ERCC-1 protein may not be useful for the prediction of tumor response in patients with unresectable colorectal cancer treated with mFOLFOX6 therapy.

  20. Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

    PubMed

    Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

    1996-09-01

    We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

  1. A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds.

    PubMed

    Gimeno-Gilles, Christine; Gervais, Marie-Laure; Planchet, Elisabeth; Satour, Pascale; Limami, Anis M; Lelievre, Eric

    2011-03-01

    MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a down-regulated gene by suppressive subtractive hybridization between two mRNA populations of embryo axes harvested before and after radicle emergence. MtSAP1 is the first gene encoding a SAP with A20 and AN1 zinc-finger domains characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in particular a strong homology in the A20 and AN1 conserved domains. MtSAP1 gene expression increased in the embryos during the acquisition of tolerance to desiccation, reached its maximum in dry seed and decreased dramatically during the first hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid and desiccation treatments induced MtSAP1 gene expression and protein accumulation in embryo axis, while mild drought stress did not affect significantly its expression. This profile of expression along with the presence of anaerobic response elements and ABRE sequences in the upstream region of the gene is consistent with a role of MtSAP1 in the tolerance of low oxygen availability and desiccation during late stages of seed maturation. Silencing of MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein is required for adequate accumulation of storage globulin proteins, vicilin and legumin, and for the development of embryos able to achieve successful germination. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  2. Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

    PubMed Central

    Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

    2013-01-01

    Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

  3. A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

    PubMed

    Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

    2013-02-01

    Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

  4. Expression of Spindle and Kinetochore-Associated Protein 1 Is Associated with Poor Prognosis in Papillary Thyroid Carcinoma

    PubMed Central

    Dong, Chao; Wang, Xiao-li; Ma, Bin-lin

    2015-01-01

    Aim. Spindle and kinetochore-associated protein 1 (SKA1) is one subtype of SKA, whose protein can make spindle microtubules attach steadily to the kinetochore in the middle of mitosis. At present, there are fewer researches on the relationship between SKA1 expression and tumor development. Methods. In this study, immunohistochemical analysis was used to determine the expression of SKA1 in papillary thyroid carcinoma (PTC) and adjacent tissues. We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis to further verify the results. Results. We found that SKA1 expression was significantly higher in PTC tissues than normal adjacent tissues (P < 0.05). There existed a significant correlation among a higher SKA1 expression, including lymphoid node (P = 0.005), clinical stage (P = 0.015), and extrathyroid invasion (P = 0.004). Survival analysis showed high SKA1 expression in PTC patients more likely to relapse after surgery. Conclusion. High SKA1 expression is predictive of poor prognosis of PTC, implying that SKA1 may be a promising new target for targeted therapies for PTC. PMID:26063960

  5. Citrullinated Chemokines in Rheumatoid Arthritis

    DTIC Science & Technology

    2016-12-01

    immunosorbent assay ( ELISA ) in RA and normal (NL) sera and in RA, osteoarthritis (OA), and other inflammatory rheumatic disease (OD) synovial fluids (SFs... ELISA ) Epithelial Neutrophil Chemoattractant Peptide-78 (ENA-78/CXCL5) Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) Macrophage Inflammatory...Citrullinated chemokines are highly expressed in RA sera and SFs. Citrullinated chemokines were measured using an ELISA in which chemokines were captured on

  6. Sequence variations and protein expression levels of the two immune evasion proteins Gpm1 and Pra1 influence virulence of clinical Candida albicans isolates.

    PubMed

    Luo, Shanshan; Hipler, Uta-Christina; Münzberg, Christin; Skerka, Christine; Zipfel, Peter F

    2015-01-01

    Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence.

  7. Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms.

    PubMed

    Guan, Hexin; Zheng, Zhengui; Grey, Paris H; Li, Yuhua; Oppenheimer, David G

    2011-05-01

    Floral transition is a critical and strictly regulated developmental process in plants. Mutations in Arabidopsis LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1)/TERMINAL FLOWER 2 (TFL2) result in early and terminal flowers. Little is known about the gene expression, function and evolution of plant LHP1 homologs, except for Arabidopsis LHP1. In this study, the conservation and divergence of plant LHP1 protein sequences was analyzed by sequence alignments and phylogeny. LHP1 expression patterns were compared among taxa that occupy pivotal phylogenetic positions. Several relatively conserved new motifs/regions were identified among LHP1 homologs. Phylogeny of plant LHP1 proteins agreed with established angiosperm relationships. In situ hybridization unveiled conserved expression of plant LHP1 in the axillary bud/tiller, vascular bundles, developing stamens, and carpels. Unlike AtLHP1, cucumber CsLHP1-2, sugarcane SoLHP1 and maize ZmLHP1, rice OsLHP1 is not expressed in the shoot apical meristem (SAM) and the OsLHP1 transcript level is consistently low in shoots. "Unequal crossover" might have contributed to the divergence in the N-terminal and hinge region lengths of LHP1 homologs. We propose an "insertion-deletion" model for soybean (Glycine max L.) GmLHP1s evolution. Plant LHP1 homologs are more conserved than previously expected, and may favor vegetative meristem identity and primordia formation. OsLHP1 may not function in rice SAM during floral induction.

  8. [Correlation of chromosome 1p and 19q status and expression of R132H mutant IDH1 protein in oligodendroglial tumors].

    PubMed

    Yao, Kun; Duan, Zejun; Hu, Zeliang; Bian, Yu; Qi, Xueling

    2014-10-01

    To correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors. The study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion. Deletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05). A significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.

  9. Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12.

    PubMed

    Wobus, Manja; List, Catrin; Dittrich, Tobias; Dhawan, Abhishek; Duryagina, Regina; Arabanian, Laleh S; Kast, Karin; Wimberger, Pauline; Stiehler, Maik; Hofbauer, Lorenz C; Jakob, Franz; Ehninger, Gerhard; Anastassiadis, Konstantinos; Bornhäuser, Martin

    2015-01-01

    We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches. © 2014 UICC.

  10. Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

    PubMed Central

    Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

    2011-01-01

    Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

  11. Expression of fibroblast specific protein-1 in pleural tuberculosis and its clinical biological significance

    PubMed Central

    2014-01-01

    Background Fibroblast specific protein-1 (S100A4) is related with many fibrotic diseases, but its role in the pathogenesis of pleural fibrosis has not been fully elucidated. Then we aim to investigate the expression and effect of fibroblast specific protein-1 (S100A4) in pleural tuberculosis and, subsequently, pleural fibrosis. Methods The expression of S100A4 in pleura was examined in 30 patients with pleural tuberculosis and 5 control (disease-free) patients by immunohistochemistry using the streptavidin-peroxidase (S-P) conjugated method. Results The expression of S100A4 in pleura was mainly distributed in the nucleus and cytoplasm of fibroblasts and vascular endothelial cells, and the positive rate was 90.0% (27 out of 30 patients with pleural tuberculosis). There were no expressions of S100A4 in the control group. In the pleura of all 30 patients with pleural tuberculosis, S100A4 had a higher expression in the two- to eight-week duration of the disease. Conclusions S100A4 plays an important role in the phenotypic transformation of pleural mesothelial cells and the development of pleural fibrosis. PMID:24885536

  12. Elevated transcription factor specificity protein 1 in autistic brains alters the expression of autism candidate genes.

    PubMed

    Thanseem, Ismail; Anitha, Ayyappan; Nakamura, Kazuhiko; Suda, Shiro; Iwata, Keiko; Matsuzaki, Hideo; Ohtsubo, Masafumi; Ueki, Takatoshi; Katayama, Taiichi; Iwata, Yasuhide; Suzuki, Katsuaki; Minoshima, Shinsei; Mori, Norio

    2012-03-01

    Profound changes in gene expression can result from abnormalities in the concentrations of sequence-specific transcription factors like specificity protein 1 (Sp1). Specificity protein 1 binding sites have been reported in the promoter regions of several genes implicated in autism. We hypothesize that dysfunction of Sp1 could affect the expression of multiple autism candidate genes, contributing to the heterogeneity of autism. We assessed any alterations in the expression of Sp1 and that of autism candidate genes in the postmortem brain (anterior cingulate gyrus [ACG], motor cortex, and thalamus) of autism patients (n = 8) compared with healthy control subjects (n = 13). Alterations in the expression of candidate genes upon Sp1/DNA binding inhibition with mithramycin and Sp1 silencing by RNAi were studied in SK-N-SH neuronal cells. We observed elevated expression of Sp1 in ACG of autism patients (p = .010). We also observed altered expression of several autism candidate genes. GABRB3, RELN, and HTR2A showed reduced expression, whereas CD38, ITGB3, MAOA, MECP2, OXTR, and PTEN showed elevated expression in autism. In SK-N-SH cells, OXTR, PTEN, and RELN showed reduced expression upon Sp1/DNA binding inhibition and Sp1 silencing. The RNA integrity number was not available for any of the samples. Transcription factor Sp1 is dysfunctional in the ACG of autistic brain. Consequently, the expression of potential autism candidate genes regulated by Sp1, especially OXTR and PTEN, could be affected. The diverse downstream pathways mediated by the Sp1-regulated genes, along with the environmental and intracellular signal-related regulation of Sp1, could explain the complex phenotypes associated with autism.

  13. Epac1, PDE4, and PKC protein expression and their association with AKAP95, Cx43, and cyclinD2/E1 in breast cancer tissues.

    PubMed

    Huang, Ping; Sun, Qian; Zhuang, Wenxin; Peng, Kuan; Wang, Dai; Yao, Youliang; Guo, Dongbei; Zhang, Lu; Shen, Chuhan; Sun, Mengyun; Tang, Chaoying; Teng, Bogang; Zhang, Yongxing

    2017-09-01

    This study was conducted to investigate the exchange protein directly activated by cAMP (Epac1), PDE4, and PKC expression in breast cancer tissues, and the correlation between these proteins and AKAP95, Cx43, cyclin D2, and cyclin E1. PV-9000 two-step immunohistochemistry was used to analyze protein expression. The positive rate of Epac1 protein expression in breast cancer tissues (58%) was higher than in para-carcinoma tissues (10%) (P < 0.05). There were no significant differences in the positive rates of PDE4 and PKC expression between breast cancer and para-carcinoma tissues (P > 0.05). The positive expression rate of PDE4 was higher in the P53 protein positive group compared to the P53 negative group (P < 0.05). Correlations between Epac1 and cyclin D2, PDE4 and cyclin D2, AKAP95 and PKC, Cx43 and PKC, and cyclin D2 and PKC proteins were observed (P < 0.05). Epac1 expression in breast cancer tissues was increased, suggesting that the protein may be involved in the development of breast cancer. Correlations between Epac1 and cyclin D2, PDE4 and cyclin D2, AKAP95 and PKC, Cx43 and PKC, and cyclin D2 and PKC proteins suggested synergistic effects among these proteins in the development of breast cancer. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  14. Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease

    PubMed Central

    Uribe, Mary Luz; Haro, Carmen; Campello, Laura; Cruces, Jesús; Martín-Nieto, José

    2016-01-01

    Purpose The POMGNT1 gene, encoding protein O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene’s lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. Methods Using reverse transcription (RT)–PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. Results POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. Conclusions

  15. Differentially co-expressed interacting protein pairs discriminate samples under distinct stages of HIV type 1 infection.

    PubMed

    Yoon, Dukyong; Kim, Hyosil; Suh-Kim, Haeyoung; Park, Rae Woong; Lee, KiYoung

    2011-01-01

    Microarray analyses based on differentially expressed genes (DEGs) have been widely used to distinguish samples across different cellular conditions. However, studies based on DEGs have not been able to clearly determine significant differences between samples of pathophysiologically similar HIV-1 stages, e.g., between acute and chronic progressive (or AIDS) or between uninfected and clinically latent stages. We here suggest a novel approach to allow such discrimination based on stage-specific genetic features of HIV-1 infection. Our approach is based on co-expression changes of genes known to interact. The method can identify a genetic signature for a single sample as contrasted with existing protein-protein-based analyses with correlational designs. Our approach distinguishes each sample using differentially co-expressed interacting protein pairs (DEPs) based on co-expression scores of individual interacting pairs within a sample. The co-expression score has positive value if two genes in a sample are simultaneously up-regulated or down-regulated. And the score has higher absolute value if expression-changing ratios are similar between the two genes. We compared characteristics of DEPs with that of DEGs by evaluating their usefulness in separation of HIV-1 stage. And we identified DEP-based network-modules and their gene-ontology enrichment to find out the HIV-1 stage-specific gene signature. Based on the DEP approach, we observed clear separation among samples from distinct HIV-1 stages using clustering and principal component analyses. Moreover, the discrimination power of DEPs on the samples (70-100% accuracy) was much higher than that of DEGs (35-45%) using several well-known classifiers. DEP-based network analysis also revealed the HIV-1 stage-specific network modules; the main biological processes were related to "translation," "RNA splicing," "mRNA, RNA, and nucleic acid transport," and "DNA metabolism." Through the HIV-1 stage-related modules, changing

  16. Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

    PubMed Central

    Harris, Deshea L.

    2007-01-01

    Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

  17. Does the Oral Contraceptive Pill Increase Plasma Intercellular Adhesion Molecule-1, Monocyte Chemoattractant Protein-1, and Tumor Necrosis Factor-α Levels in Women with Polycystic Ovary Syndrome: A Pilot Study.

    PubMed

    Yousuf, Syed Douhath; Rashid, Fouzia; Mattoo, Tahira; Shekhar, Chander; Mudassar, Syed; Zargar, M Afzal; Ganie, Mohd Ashraf

    2017-02-01

    Polycystic ovary syndrome (PCOS), the most common endocrinopathy of women, is a state of chronic low-grade inflammation and is closely linked to type 2 diabetes mellitus and cardiovascular disease. Oral contraceptive pills (OCPs), is the usual first choice of treatment in women with PCOS. Because OCP use has been linked to the risk of venous thrombosis and there are limited data on the effect of OCP use on the inflammatory state of women with PCOS, our objective was to compare the levels of intercellular adhesion molecule (ICAM)-1, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 between drug-naive and OCP-treated women with PCOS. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Consequent to women diagnosed with PCOS on the basis of Rotterdam 2003 criteria, either treated with OCPs (ethinylestradiol 0.03 mg, levonogestrel-0.15 mg) for a period of 6 months (n = 50) or drug-naive (n = 51) were enrolled in this cross-sectional study. The mean ages of patients and control participants were comparable (21.99 ± 4.78 vs 21.92 ± 5.83 years; P = .947) as was body mass index (24.47 ± 3.92 vs 23.66 ± 3.43; P = .271). Clinical and androgen excess symptoms were significantly better in the OCP group compared with the drug-naive group (P = .01, P = .04). Total cholesterol and low-density lipoprotein cholesterol levels were significantly higher in the OCP group (P = .01). Plasma ICAM-1 levels, TNF-α levels, and MCP-1 levels showed a higher trend in patients but reached statistical significance only in cases of ICAM-1 and TNF-α (P = .01). OCP treatment of 6 months increases plasma ICAM-1, MCP-1, and TNF-α levels among women with PCOS, although OCPs significantly help in ameliorating features of hyperandrogenism and regularizing menstrual cycles. These cytokines correlate positively with many metabolic parameters including plasma glucose, lipids, and homeostatic model assessment

  18. Macrophage inflammatory protein 1 alpha expression by synovial fluid neutrophils in rheumatoid arthritis

    PubMed Central

    Hatano, Y.; Kasama, T.; Iwabuchi, H.; Hanaoka, R.; Takeuchi, H.; Jing, L.; Mori, Y.; Kobayashi, K.; Negishi, M.; Ide, H.; Adachi, M.

    1999-01-01

    OBJECTIVE—To determine the contribution made by synovial fluid (SF) neutrophils to the augmented expression of macrophage inflammatory protein 1 α (MIP-1α) in rheumatoid arthritis (RA).
METHODS—Neutrophils were isolated from samples of SF from RA patients and peripheral blood (PB) samples from RA patients and healthy controls. Cell associated MIP-1α was visualised immunohistochemically, and cell associated MIP-1α as well as MIP-1α secreted into the SF was assayed by ELISA. Steady state expression of MIP-1α mRNA was assessed by reverse transcription polymerase chain reaction (RT-PCR).
RESULTS—Freshly isolated SF neutrophils contained significantly higher concentrations of both MIP-1α protein and its transcript than PB neutrophils from either RA patients or healthy controls; incubation in the absence or presence of tumour necrosis factor α for 24 hours resulted in a significant increase in MIP-1α secretion by RA SF neutrophils compared with neutrophils obtained from either normal PB or RA PB; and expression of MIP-1α by SF neutrophils was well correlated with both RA disease activity and SF mononuclear cell (MNC) counts.
CONCLUSION—Expression and secretion of MIP-1α by SF neutrophils may be indicative of local and systemic inflammation in RA. Moreover, this C-C chemokine may contribute to the recruitment of MNCs from the bloodstream into synovial joints and tissues.

 PMID:10225815

  19. Specific Distribution of the Autophagic Protein GABARAPL1/GEC1 in the Developing and Adult Mouse Brain and Identification of Neuronal Populations Expressing GABARAPL1/GEC1

    PubMed Central

    Le Grand, Jaclyn Nicole; Bon, Karine; Fraichard, Annick; Zhang, Jianhua; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël; Delage-Mourroux, Régis

    2013-01-01

    Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain. PMID:23690988

  20. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

  1. TPS1 terminator increases mRNA and protein yield in a Saccharomyces cerevisiae expression system.

    PubMed

    Yamanishi, Mamoru; Katahira, Satoshi; Matsuyama, Takashi

    2011-01-01

    Both terminators and promoters regulate gene expression. In Saccharomyces cerevisiae, the TPS1 terminator (TPS1t), coupled to a gene encoding a fluorescent protein, produced more transgenic mRNA and protein than did similar constructs containing other terminators, such as CYC1t, TDH3t, and PGK1t. This suggests that TPS1t can be used as a general terminator in the development of metabolically engineered yeast in high-yield systems.

  2. Epigenetic Induction of EGR-1 Expression by the Amyloid Precursor Protein during Exposure to Novelty

    PubMed Central

    Hendrickx, Aurélie; Pierrot, Nathalie; Tasiaux, Bernadette; Schakman, Olivier; Brion, Jean-Pierre; Kienlen-Campard, Pascal; De Smet, Charles; Octave, Jean-Noël

    2013-01-01

    Following transcriptome comparison of primary cultures isolated from brain of mice expressing or not the amyloid precursor protein APP, we found transcription of the EGR-1 gene to be regulated by APP. In primary cultures of cortical neurons, APP significantly down regulated EGR-1 expression at both mRNA and protein levels in a γ-secretase independent manner. The intracellular domain of APP did not interact with EGR-1 gene promoter, but enrichment of acetylated histone H4 at the EGR-1 promoter region was measured in APP-/- neurons, as well as in brain of APP-/- mice, in which increase in EGR-1 expression was also measured. These results argue for an important function of APP in the epigenetic regulation of EGR-1 gene transcription both in vitro and in vivo. In APP-/- mice, constitutive overexpression of EGR-1 in brain impaired epigenetic induction of this early transcriptional regulator during exposure to novelty. Altogether, these results indicate an important function of APP in the epigenetic regulation of the transcription of EGR-1, known to be important for memory formation. PMID:24066134

  3. Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

    PubMed

    Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

    2010-10-15

    The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

  4. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  5. Sperm navigation along helical paths in 3D chemoattractant landscapes

    PubMed Central

    Jikeli, Jan F.; Alvarez, Luis; Friedrich, Benjamin M.; Wilson, Laurence G.; Pascal, René; Colin, Remy; Pichlo, Magdalena; Rennhack, Andreas; Brenker, Christoph; Kaupp, U. Benjamin

    2015-01-01

    Sperm require a sense of direction to locate the egg for fertilization. They follow gradients of chemical and physical cues provided by the egg or the oviduct. However, the principles underlying three-dimensional (3D) navigation in chemical landscapes are unknown. Here using holographic microscopy and optochemical techniques, we track sea urchin sperm navigating in 3D chemoattractant gradients. Sperm sense gradients on two timescales, which produces two different steering responses. A periodic component, resulting from the helical swimming, gradually aligns the helix towards the gradient. When incremental path corrections fail and sperm get off course, a sharp turning manoeuvre puts sperm back on track. Turning results from an ‘off' Ca2+ response signifying a chemoattractant stimulation decrease and, thereby, a drop in cyclic GMP concentration and membrane voltage. These findings highlight the computational sophistication by which sperm sample gradients for deterministic klinotaxis. We provide a conceptual and technical framework for studying microswimmers in 3D chemical landscapes. PMID:26278469

  6. Sperm navigation along helical paths in 3D chemoattractant landscapes.

    PubMed

    Jikeli, Jan F; Alvarez, Luis; Friedrich, Benjamin M; Wilson, Laurence G; Pascal, René; Colin, Remy; Pichlo, Magdalena; Rennhack, Andreas; Brenker, Christoph; Kaupp, U Benjamin

    2015-08-17

    Sperm require a sense of direction to locate the egg for fertilization. They follow gradients of chemical and physical cues provided by the egg or the oviduct. However, the principles underlying three-dimensional (3D) navigation in chemical landscapes are unknown. Here using holographic microscopy and optochemical techniques, we track sea urchin sperm navigating in 3D chemoattractant gradients. Sperm sense gradients on two timescales, which produces two different steering responses. A periodic component, resulting from the helical swimming, gradually aligns the helix towards the gradient. When incremental path corrections fail and sperm get off course, a sharp turning manoeuvre puts sperm back on track. Turning results from an 'off' Ca(2+) response signifying a chemoattractant stimulation decrease and, thereby, a drop in cyclic GMP concentration and membrane voltage. These findings highlight the computational sophistication by which sperm sample gradients for deterministic klinotaxis. We provide a conceptual and technical framework for studying microswimmers in 3D chemical landscapes.

  7. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP.

    PubMed

    Page, Stephen H; Wright, Edward K; Gama, Lucio; Clements, Janice E

    2011-01-01

    CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

  8. Expression of multidrug resistance-related protein (MRP-1), lung resistance-related protein (LRP) and topoisomerase-II (TOPO-II) in Wilms' tumor: immunohistochemical study using TMA methodology.

    PubMed

    Fridman, Eduard; Skarda, Jozef; Pinthus, Jonatan H; Ramon, Jonathan; Mor, Yoran

    2008-06-01

    MRP-1, LRP and TOPO-II are all associated with protection of the cells from the adverse effects of various chemotherapeutics. The aim of this study was to measure the expression of these proteins in Wilms' tumor (WT). TMA block was constructed from 14 samples of WT's and from xenografts derived from them. Sections of the TMA were used for immunostaining against MRP-1, LRP and TOPO-IIa. All normal kidneys expressed MRP-1 but were either weakly or negatively stained for LRP and TOPO-IIa. In WT samples, MRP-1 was universally expressed, exclusively in the tubular component, while there was no expression of LRP and TOPO-IIa showed heterogeneous distribution. The xenografts varied in their MRP-1 and TOPO-IIa expression and exhibited weak/negative staining of LRP. This study shows that although all the proteins evaluated, had different expression patterns in the tumor samples, the most prominent changes in expression were found for MRP-1. The exact clinical implications of these changes in expression and their relevance to the resistance of these tumors to chemotherapy requires further investigation. The finding of different expression profiles for the multidrug resistance proteins in the original WT's and their xenografts suggests that the results of animal cancer models may be difficult to interpret.

  9. Sulforaphane inhibits the interferon-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 pancreatic β-cells through the downregulation of IRF-1, STAT-1 and PKB.

    PubMed

    Park, Yu-Kyoung; Ramalingam, Mahesh; Kim, Shin; Jang, Byeong-Churl; Park, Jong Wook

    2017-09-01

    Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic β-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible T‑cell alpha chemoattractant (I-TAC), in INS‑1 cells, a rat pancreatic β-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS‑1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS‑1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS‑1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS‑1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFN‑γ‑induced expressions of MIG and I-TAC, but not IP-10, in INS‑1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin

  10. Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype

    PubMed Central

    Liu, Qin; Collin, Rob W. J.; Cremers, Frans P. M.; den Hollander, Anneke I.; van den Born, L. Ingeborgh; Pierce, Eric A.

    2012-01-01

    Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3rd exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled. PMID:22927954

  11. A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3

    PubMed Central

    Liu, Ben; Zhong, Mintao; Lun, Yongzhi; Wang, Xiaoli; Sun, Wenchang; Li, Xingyun; Ning, Anhong; Cao, Jing; Zhang, Wei; Liu, Lei; Huang, Min

    2012-01-01

    An apoptosis correlated molecule—protein Latcripin-1 of Lentinula edodes C91–3—was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C91–3. According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3′-Full Rapid Amplification of cDNA Ends (RACE) and 5′-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins. PMID:22754362

  12. Atractylenolide I restores HO-1 expression and inhibits Ox-LDL-induced VSMCs proliferation, migration and inflammatory responses in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Weifeng, E-mail: liwf@mail.xjtu.edu.cn; Zhi, Wenbing; Liu, Fang

    Pathogenesis of atherosclerosis is characterized by the proliferation and migration of vascular smooth muscle cells (VSMCs) and inflammatory lesions. The aim of this study is to elucidate the effect of atractylenolide I (AO-I) on smooth muscle cell inflammation, proliferation and migration induced by oxidized modified low density lipoprotein (Ox-LDL). Here, We found that atractylenolide I inhibited Ox-LDL-induced VSMCs proliferation and migration in a dose-dependent manner, and decreased the production of inflammatory cytokines and the expression of monocyte chemoattractant protein-1 (MCP-1) in VSMCs. The study also identified that AO-I prominently inhibited p38-MAPK and NF-κB activation. More importantly, the specific heme oxygenase-1more » (HO-1) inhibitor zinc protoporphyrin (ZnPP) IX partially abolished the beneficial effects of atractylenolide I on Ox-LDL-induced VSMCs. Furthermore, atractylenolide I blocked the foam cell formation in macrophages induced by Ox-LDL. In summary, inhibitory roles of AO-I in VSMCs proliferation and migration, lipid peroxidation and subsequent inflammatory responses might contribute to the anti-atherosclerotic property of AO-I. - Highlights: • AO-I inhibited Ox-LDL-induced VSMCs proliferation and migration. • AO-I alleviated inflammatory response via inhibiting TNF-α, IL-6 and NO production. • AO-I restored HO-1 expression and down-regulated PCNA expression. • MCP-1 overexpression is potentially regulated by NF-κB and p38 MAPK pathway. • AO-I possesses strong anti-lipid peroxidation effect.« less

  13. Isthmin 1 Is a Secreted Protein Expressed in Skin, Mucosal Tissues, and NK, NKT, and Th17 Cells

    PubMed Central

    Valle-Rios, Ricardo; Maravillas-Montero, José L.; Burkhardt, Amanda M.; Martinez, Cynthia; Buhren, Bettina Alexandra; Homey, Bernhard; Gerber, Peter Arne; Robinson, Octavio; Hevezi, Peter

    2014-01-01

    Using a comprehensive microarray database of human gene expression, we identified that in mammals, a secreted protein known as isthmin 1 (ISM1) is expressed in skin, mucosal tissues, and selected lymphocyte populations. ISM1 was originally identified in Xenopus brain during development, and it encodes a predicted ∼50-kDa protein containing a signal peptide, a thrombospondin domain, and an adhesion-associated domain. We confirmed the pattern of expression of ISM1 in both human and mouse tissues. ISM1 is expressed by DX5+ lung lymphocytes that include NK and NKT-like cells, and is also expressed by some CD4+ T cells upon activation but its expression increases significantly when CD4+ T cells were polarized to the Th17 lineage in vitro. The presence of IFN-γ during CD4+ T cell polarization inhibits ISM1 expression. Given that ISM1 has been reported to have anti-angiogenic properties, these observations suggest that ISM1 is a mediator of lymphocyte effector functions and may participate in both innate and acquired immune responses. PMID:24956034

  14. Isthmin 1 is a secreted protein expressed in skin, mucosal tissues, and NK, NKT, and th17 cells.

    PubMed

    Valle-Rios, Ricardo; Maravillas-Montero, José L; Burkhardt, Amanda M; Martinez, Cynthia; Buhren, Bettina Alexandra; Homey, Bernhard; Gerber, Peter Arne; Robinson, Octavio; Hevezi, Peter; Zlotnik, Albert

    2014-10-01

    Using a comprehensive microarray database of human gene expression, we identified that in mammals, a secreted protein known as isthmin 1 (ISM1) is expressed in skin, mucosal tissues, and selected lymphocyte populations. ISM1 was originally identified in Xenopus brain during development, and it encodes a predicted ∼50-kDa protein containing a signal peptide, a thrombospondin domain, and an adhesion-associated domain. We confirmed the pattern of expression of ISM1 in both human and mouse tissues. ISM1 is expressed by DX5(+) lung lymphocytes that include NK and NKT-like cells, and is also expressed by some CD4(+) T cells upon activation but its expression increases significantly when CD4(+) T cells were polarized to the Th17 lineage in vitro. The presence of IFN-γ during CD4(+) T cell polarization inhibits ISM1 expression. Given that ISM1 has been reported to have anti-angiogenic properties, these observations suggest that ISM1 is a mediator of lymphocyte effector functions and may participate in both innate and acquired immune responses.

  15. Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines

    PubMed Central

    Miura, Tanya A.; Wang, Jieru; Holmes, Kathryn V.; Mason, Robert J.

    2007-01-01

    We analyzed the ability of two rat coronavirus (RCoV) strains, sialodacryoadenitis virus (SDAV) and Parker’s RCoV (RCoV-P), to infect rat alveolar type I cells and induce chemokine expression. Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCoV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemoattractants. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation. PMID:17804032

  16. Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

    PubMed Central

    Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

    2013-01-01

    Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. PMID:24376827

  17. Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

    PubMed

    Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

    2013-01-01

    Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

  18. Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

    PubMed Central

    WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

    2013-01-01

    This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

  19. Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

    PubMed Central

    Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

    2005-01-01

    Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

  20. [Extranodal nasal type NK/T-cell lymphoma: the expression of Epstein-Barr virus latent membrane protein 1 and its significance of prognosis].

    PubMed

    Zhao, Sha; Liu, Wei-ping; Zhang, Wen-yan; Li, Gan-di

    2005-05-01

    To investigate the expression and prognostic significance of Epstein-Barr virus latent membrane protein 1 in extranodal nasal type NK/T-cell lymphoma in the Chengdu area. The expression of latent membrane protein-1 (LMP1) was detected by immunohistochemistry (IHC) and DNA-PCR in 67 cases of extranodal nasal type NK/T-cell lymphoma, and the differences in survival rate between positive and negative expression groups of LMP1-protien and LMP1-DNA were analyzed respectively. Ten (14.93%) cases were positive at LMP1-protein level, and fifty-six (83.58%) were positive at LMP1-DNA level. The total expression rate of LMP1 was 83.58%. No statistically significant difference was observed between the expression of LMP1 and prognosis (P = 0.678) and between the expression of LMP1-DNA and prognosis (P = 0.943). LMP1 was shown to be closely associated with extranodal nasal type NK/T-cell lymphoma in Chengdu. The expression rate of LMP1 at protein level was different from that at DNA level. No relationship was found between the prognosis and the LMP1 expression in extranodal nasal type NK/T-cell lymphoma.

  1. Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

    PubMed

    Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

    2018-05-11

    Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

  2. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  3. Biphasic and bilateral changes in striatal VGLUT1 and 2 protein expression in hemi-Parkinson rats.

    PubMed

    Massie, Ann; Schallier, Anneleen; Vermoesen, Katia; Arckens, Lutgarde; Michotte, Yvette

    2010-09-01

    Parkinson's disease is characterized by disturbed glutamatergic neurotransmission in the striatum. Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1 and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na(+)/K(+)-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2 transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time intervals post unilateral 6-OHDA injection into the medial forebrain bundle versus controls. Three weeks post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2 expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-Parkinson rats whereas the striatal expression of VGLUT2 was comparable to control rats. No effect could be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic neurotransmission from the two types of striatal afferents. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion.

    PubMed

    Kucia, Magda; Jankowski, Kacper; Reca, Ryan; Wysoczynski, Marcin; Bandura, Laura; Allendorf, Daniel J; Zhang, Jin; Ratajczak, Janina; Ratajczak, Mariusz Z

    2004-03-01

    Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine-chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an alpha-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1-CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1-CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential

  5. Kindler syndrome protein Kindlin-1 is mainly expressed in adult tissues originating from ectoderm/endoderm.

    PubMed

    Zhan, Jun; Yang, Mei; Zhang, Jing; Guo, YongQing; Liu, Wei; Zhang, HongQuan

    2015-05-01

    Mutations of integrin-interacting protein Kindlin-1 cause Kindler syndrome and deregulation of Kindlin-1 is implicated in human cancers. The Kindlin-1-related diseases are confined in limited tissue types. However, Kindlin-1 tissue distribution and the dogma that governs Kindlin-1 expression in normal human body are elusive. This study examined Kindlin-1 expression in normal human adult organs, human and mouse embryonic organs by immunohistochemical analyses. We identified a general principle that the level of Kindlin-1 expression in tissues is tightly correlated with the corresponding germ layers from which these tissues originate. We compared the expression of Kindlin-1 with Kindlin-2 and found that Kindlin-1 is highly expressed in epithelial tissues derived from ectoderm and endoderm, whereas Kindlin-2 is mainly expressed in mesoderm-derived tissues. Likewise, Kindlin-1 was also found highly expressed in endoderm/ectoderm-derived tissues in human and mouse embryos. Our findings indicate that Kindlin-1 may play an importance role in the development of endoderm/ectoderm related tissues.

  6. AR-v7 protein expression is regulated by protein kinase and phosphatase

    PubMed Central

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  7. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

    PubMed

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-02-15

    To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

  8. Expression of Small Heat-Shock Proteins at Low Temperatures1

    PubMed Central

    Sabehat, Adnan; Lurie, Susan; Weiss, David

    1998-01-01

    We previously reported that short exposure of tomato (Lycopersicon esculentum L.) fruits to high temperature protects them from chilling injury. To study the involvement of heat-shock proteins (HSPs) in the acquisition of low-temperature tolerance, we cloned two heat-shock-induced genes that are also expressed at low temperatures. The cloned cDNAs belong to the small HSP group. Sequence analyses of the clones showed perfect homology to the tomato-ripening gene tom66 and to the tomato chloroplastic HSP21 gene tom111. The expression of both genes was induced by high temperature in fruits, flowers, leaves, and stems, but not by low or ambient temperatures or by other stresses such as drought and anaerobic conditions. When the heated fruits were transferred to low temperature, tom66 and tom111 mRNA levels first decreased but were then reinduced. Induction was not observed in nonheated fruits at low temperature. Immunodetection of tom111-encoded protein indicated that this protein is present at low temperatures in the heated fruits. The results of this study show that the expression of tom66 and tom111 is correlated with protection against some, but not all, symptoms of chilling injury. PMID:9625718

  9. Characterization and Expression Pattern Analysis of the T-Complex Protein-1 Zeta Subunit in Musca domestica L (Diptera).

    PubMed

    Zhao, Xuejun; Xiu, Jiangfan; Li, Yan; Ma, Huiling; Wu, Jianwei; Wang, Bo; Guo, Guo

    2017-07-01

    Chaperonins, belonging to the T-complex protein-1 (TCP-1) family, assist in the correct folding of nascent and misfolded proteins. It is well-known that in mammals, the zeta subunit of the TCP-1 complex (TCP-1ζ) plays a vital role in the folding and assembly of cytoskeleta proteins. This study reported for the first time the cloning, characterization and expression pattern analysis of the TCP-1ζ from Musca domestica, which was named as MdTCP-1ζ. The MdTCP-1ζ cDNA is 1,803 bp long with a 1,596 bp open reading frame that encodes a protein with 531 bp amino acids. The analysis of the transcriptional profile of MdTCP-1ζ using qRT-PCR revealed relatively high expression in the salivary glands and trachea at the tissues while among the developmental stages. The highest expression was observed only in the eggs suggesting that the MdTCP-1ζ may play a role in embryonic development. The expression of MdTCP-1ζ was also significantly induced after exposure to short-term heat shock and infection by Escherichia coli, Staphylococcus aureus, or Candida albicans. This suggested that MdTCP-1ζ may take part in the immune responses of housefly and perhaps contribute to the protection against cellular injury. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  10. Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

    DOE PAGES

    Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo; ...

    2017-10-07

    We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

  11. Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo

    We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured)more » longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.« less

  12. Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors

    PubMed Central

    Quakkelaar, Esther D.; Redeker, Anke; Haddad, Elias K.; Harari, Alexandre; McCaughey, Stella Mayo; Duhen, Thomas; Filali-Mouhim, Abdelali; Goulet, Jean-Philippe; Loof, Nikki M.; Ossendorp, Ferry; Perdiguero, Beatriz; Heinen, Paul; Gomez, Carmen E.; Kibler, Karen V.; Koelle, David M.; Sékaly, Rafick P.; Sallusto, Federica; Lanzavecchia, Antonio; Pantaleo, Giuseppe; Esteban, Mariano; Tartaglia, Jim; Jacobs, Bertram L.; Melief, Cornelis J. M.

    2011-01-01

    Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. PMID:21347234

  13. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Seung Heon; Kang, Sukmo; Dong, Mi Sook

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E.more » coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.« less

  14. Expression of multidrug resistance proteins in retinoblastoma

    PubMed Central

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  15. Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

    PubMed

    Park, Minhee; Lim, Jong-Sun; Lee, Hyoung-Joo; Na, Keun; Lee, Min Jung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

    2015-08-07

    Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and β-catenin, and proteins that bind β-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to β-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

  16. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  17. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  18. Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

    PubMed

    Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

    2012-01-01

    The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

  19. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 andmore » CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO

  20. Zac1, an Sp1-like protein, regulates human p21{sup WAF1/Cip1} gene expression in HeLa cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Pei-Yao; Hsieh, Tsai-Yuan; Liu, Shu-Ting

    2011-12-10

    Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21{sup WAF1/Cip1} gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity bymore » interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21{sup WAF1/Cip1} gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.« less

  1. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  2. Suppression of polyglutamine protein toxicity by co-expression of a heat-shock protein 40 and a heat-shock protein 110

    PubMed Central

    Kuo, Y; Ren, S; Lao, U; Edgar, B A; Wang, T

    2013-01-01

    A network of heat-shock proteins mediates cellular protein homeostasis, and has a fundamental role in preventing aggregation-associated neurodegenerative diseases. In a Drosophila model of polyglutamine (polyQ) disease, the HSP40 family protein, DNAJ-1, is a superior suppressor of toxicity caused by the aggregation of polyQ containing proteins. Here, we demonstrate that one specific HSP110 protein, 70 kDa heat-shock cognate protein cb (HSC70cb), interacts physically and genetically with DNAJ-1 in vivo, and that HSC70cb is necessary for DNAJ-1 to suppress polyglutamine-induced cell death in Drosophila. Expression of HSC70cb together with DNAJ-1 significantly enhanced the suppressive effects of DNAJ-1 on polyQ-induced neurodegeneration, whereas expression of HSC70cb alone did not suppress neurodegeneration in Drosophila models of either general polyQ disease or Huntington's disease. Furthermore, expression of a human HSP40, DNAJB1, together with a human HSP110, APG-1, protected cells from polyQ-induced neural degeneration in flies, whereas expression of either component alone had little effect. Our data provide a functional link between HSP40 and HSP110 in suppressing the cytotoxicity of aggregation-prone proteins, and suggest that HSP40 and HSP110 function together in protein homeostasis control. PMID:24091676

  3. Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

    PubMed

    Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

    2018-05-02

    The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

  4. Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

    PubMed

    Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

    2010-08-01

    The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

  5. Subcellular Changes in Bridging Integrator 1 Protein Expression in the Cerebral Cortex During the Progression of Alzheimer Disease Pathology.

    PubMed

    Adams, Stephanie L; Tilton, Kathy; Kozubek, James A; Seshadri, Sudha; Delalle, Ivana

    2016-08-01

    Genome-wide association studies have established BIN1 (Bridging Integrator 1) as the most significant late-onset Alzheimer disease (AD) susceptibility locus after APOE We analyzed BIN1 protein expression using automated immunohistochemistry on the hippocampal CA1 region in 19 patients with either no, mild, or moderate-to-marked AD pathology, who had been assessed by Clinical Dementia Rating and CERAD scores. We also examined the amygdala, prefrontal, temporal, and occipital regions in a subset of these patients. In non-demented controls without AD pathology, BIN1 protein was expressed in white matter, glia, particularly oligodendrocytes, and in the neuropil in which the BIN1 signal decorated axons. With increasing severity of AD, BIN1 in the CA1 region showed: 1) sustained expression in glial cells, 2) decreased areas of neuropil expression, and 3) increased cytoplasmic neuronal expression that did not correlate with neurofibrillary tangle load. In patients with AD, both the prefrontal cortex and CA1 showed a decrease in BIN1-immunoreactive (BIN1-ir) neuropil areas and increases in numbers of BIN1-ir neurons. The numbers of CA1 BIN1-ir pyramidal neurons correlated with hippocampal CERAD neuritic plaque scores; BIN1 neuropil signal was absent in neuritic plaques. Our data provide novel insight into the relationship between BIN1 protein expression and the progression of AD-associated pathology and its diagnostic hallmarks. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

  6. Cloning and expression analysis of Zmglp1, a new germin-like protein gene in maize.

    PubMed

    Fan, Zhanmin; Gu, Hongya; Chen, Xiaowei; Song, Hui; Wang, Qian; Liu, Meihua; Qu, Li-Jia; Chen, Zhangliang

    2005-06-17

    The cDNA and genomic DNA of a green tissue-specific gene were cloned from maize (Zea mays L.) using cDNA-amplified fragment length polymorphism (cDNA-AFLP) and library screening. The deduced protein was highly similar to Hordeum vulgare germin-like protein 1 (HvGLP1), and the maize gene was therefore designated Zmglp1. Northern blot specifically detected the mRNA of Zmglp1 in young whorl leaves at the early-whorl stage. However, at the late-whorl, tassel, and silk stages, Zmglp1 transcripts were highly abundant in young whorl leaves; less abundant in mature leaves, young tassels, and cobs; and not detectable in roots, immature kernels, and stalks. RNA in situ hybridization revealed that Zmglp1 expressed only in mesophyllous, phloem, and guard cells in the young whorl leaves. Deletion analysis of the promoter in transgenic Arabidopsis resulted in the identification of several regions containing important regulatory cis-elements controlling the expression levels and circadian rhythm-oscillated patterns of Zmglp1.

  7. Low expression of G protein-coupled oestrogen receptor 1 (GPER) is associated with adverse survival of breast cancer patients

    PubMed Central

    Martin, Stewart G.; Lebot, Marie N.; Sukkarn, Bhudsaban; Ball, Graham; Green, Andrew R.; Rakha, Emad A.; Ellis, Ian O.; Storr, Sarah J.

    2018-01-01

    G protein-coupled oestrogen receptor 1 (GPER), also called G protein-coupled receptor 30 (GPR30), is attracting considerable attention for its potential role in breast cancer development and progression. Activation by oestrogen (17β-oestradiol; E2) initiates short term, non-genomic, signalling events both in vitro and in vivo. Published literature on the prognostic value of GPER protein expression in breast cancer indicates that further assessment is warranted. We show, using immunohistochemistry on a large cohort of primary invasive breast cancer patients (n=1245), that low protein expression of GPER is not only significantly associated with clinicopathological and molecular features of aggressive behaviour but also significantly associated with adverse survival of breast cancer patients. Furthermore, assessment of GPER mRNA levels in the METABRIC cohort (n=1980) demonstrates that low GPER mRNA expression is significantly associated with adverse survival of breast cancer patients. Using artificial neural networks, genes associated with GPER mRNA expression were identified; these included notch-4 and jagged-1. These results support the prognostic value for determination of GPER expression in breast cancer. PMID:29899833

  8. Expression of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) in the nucleus accumbens is critical for the acquisition, expression and reinstatement of morphine-induced conditioned place preference.

    PubMed

    Lv, Xiu-Fang; Xu, Ya; Han, Ji-Sheng; Cui, Cai-Lian

    2011-09-30

    Activity-regulated cytoskeleton-associated protein (Arc), also known as activity-regulated gene 3.1 (Arg3.1), is an immediate early gene whose mRNA is selectively targeted to recently activated synaptic sites, where it is translated and enriched. This unique feature suggests a role for Arc/Arg3.1 in coupling synaptic activity to protein synthesis, leading to synaptic plasticity. Although the Arc/Arg3.1 gene has been shown to be induced by a variety of abused drugs and its protein has been implicated in diverse forms of long-term memory, relatively little is known about its role in drug-induced reward memory. In this study, we investigated the potential role of Arc/Arg3.1 protein expression in reward-related associative learning and memory using morphine-induced conditioned place preference (CPP) in rats. We found that (1) intraperitoneal (i.p.) injection of morphine (10mg/kg) increased Arc/Arg3.1 protein levels after 2h in the NAc core but not in the NAc shell. (2) In CPP experiments, Arc/Arg3.1 protein was increased in the NAc shell of rats following both morphine conditioning and the CPP expression test compared to rats that received the conditioning without the test or those that did not receive morphine conditioning. (3) Microinjection of Arc/Arg3.1 antisense oligodeoxynucleotide (AS) into the NAc core inhibited the acquisition, expression and reinstatement of morphine CPP; however, intra-NAc shell infusions of the AS only blocked the expression of CPP. These findings suggest that expression of the Arc/Arg3.1 protein in the NAc core is required for the acquisition, context-induced retrieval and reinstatement of morphine-associated reward memory, whereas Arc/Arg3.1 protein expression in the NAc shell is only critical for the context-induced retrieval of memory. As a result, Arc/Arg3.1 may be a potential therapeutic target for the prevention of drug abuse or the relapse of drug use. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. The Short Form of the Zinc Finger Antiviral Protein Inhibits Influenza A Virus Protein Expression and Is Antagonized by the Virus-Encoded NS1.

    PubMed

    Tang, Qiannan; Wang, Xinlu; Gao, Guangxia

    2017-01-15

    Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses. There are two ZAP isoforms arising from alternative splicing, which differ only at the C termini. It was recently reported that the long isoform (ZAPL) promotes proteasomal degradation of influenza A virus (IAV) proteins PA and PB2 through the C-terminal poly(ADP-ribose) polymerase (PARP) domain, which is missing in the short form (ZAPS), and that this antiviral activity is antagonized by the viral protein PB1. Here, we report that ZAP inhibits IAV protein expression in a PARP domain-independent manner. Overexpression of ZAPS inhibited the expression of PA, PB2, and neuraminidase (NA), and downregulation of the endogenous ZAPS enhanced their expression. We show that ZAPS inhibited PB2 protein expression by reducing the encoding viral mRNA levels and repressing its translation. However, downregulation of ZAPS only modestly enhanced the early stage of viral replication. We provide evidence showing that the antiviral activity of ZAPS is antagonized by the viral protein NS1. A recombinant IAV carrying an NS1 mutant that lost the ZAPS-antagonizing activity replicated better in ZAPS-deficient cells. We further provide evidence suggesting that NS1 antagonizes ZAPS by inhibiting its binding to target mRNA. These results uncover a distinct mechanism underlying the interactions between ZAP and IAV. ZAP is a host antiviral factor that has been extensively reported to inhibit the replication of certain viruses by repressing the translation and promoting the degradation of the viral mRNAs. There are two ZAP isoforms, ZAPL and ZAPS. ZAPL was recently reported to promote IAV protein degradation through the PARP domain. Whether ZAPS, which lacks the PARP domain, inhibits IAV and the underlying mechanisms remained to be determined. Here, we show that ZAPS posttranscriptionally inhibits IAV protein expression. This antiviral activity of ZAP is antagonized by the viral

  10. Suppression of methylmercury-induced MIP-2 expression by N-acetyl-L-cysteine in murine RAW264.7 macrophage cell line.

    PubMed

    David, Juliet; Nandakumar, Athira; Muniroh, Muflihatul; Akiba, Suminori; Yamamoto, Megumi; Koriyama, Chihaya

    2017-11-09

    The aim of this study is to examine the inflammatory-cytokine expressions in the presence of non-cytotoxic dose of methylmercury (MeHg) in murine macrophages, which is suspected to play an important role in brain damage caused by MeHg exposure. We focused on murine macrophage inflammatory protein-2 (MIP-2), keratinocyte chemoattractant (KC), and monocyte chemoattractant protein-5 (MCP-5). MIP-2 and KC are murine functional homologues of human IL-8 and MCP-5 for human MCP-1. Furthermore, we examined the suppressive effect of N-acetyl-L-cysteine (NAC) on the MeHg-induced inflammatory cytokines. In a murine RAW264.7 macrophage cell line, MeHg-induced cytokine expressions were measured using real-time PCR. The suppressive effect of NAC was examined by putting it into the culture medium together with MeHg (co-treatment). In addition, pre- and post-treatment experiments were conducted, in which the cells were treated with NAC before and after MeHg exposure, respectively. Exposure to a non-cytotoxic dose of MeHg up-regulated the mRNA expression of MIP-2 and MCP-5. On the other hand, KC expression was not induced in the presence of MeHg. Effect of MeHg on MIP-2 expressions was suppressed by pre-, co-, and post-treatment with NAC. However, the suppressive effect of pre-treatment was less than the post-treatment, which was as effective as co-treatment. In functional homologues of human IL-8, only MIP-2 expression, not KC, was activated in the presence of non-cytotoxic dose of MeHg in murine RAW264.7 macrophage cell line. The more evident inhibitory effect of NAC observed in post-treatment experiments suggests a possible involvement of intracellular activities such as antioxidant effects.

  11. Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma.

    PubMed

    Duś-Szachniewicz, Kamila; Woźniak, Marta; Nelke, Kamil; Gamian, Elżbieta; Gerber, Hanna; Ziółkowski, Piotr

    2015-12-01

    The actions of tyrosine phosphorylation and dephosphorylation are controlled by tyrosine kinases and phosphatases. Although substantial previous data have revealed the role of several protein tyrosine phosphatases (PTPs) in various cancers, the function of protein tyrosine phosphatase receptor R (PTPRR) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) proteins in oral cavity squamous cell carcinoma (SCC) has not been studied to date. The PTPRR and PTPRZ1 immunoreactivity in 67 formalin-fixed and paraffin-embedded oral cancer tissues at different stages were analyzed with the technique of immunohistochemistry (IHC). The presence of PTPRR in cancerous tissue was confirmed by Western blotting. The occurrence of PTPRR and PTPRZ1 proteins in the cancer specimens was more frequent in lower grade tumors. In addition, the association between the immunoreactivity of both examined proteins and improved patients survival was detected. Moreover, the PTPRR expression was found to be related to the absence of synchronous lymph node involvement. The above results indicate that the PTPRR and PTPRZ1 protein expression should be monitored in oral cancer for patients' prognostic stratification. © 2015 Wiley Periodicals, Inc.

  12. Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: prediction and validation.

    PubMed

    Datta, Moumita; Choudhury, Ananyo; Lahiri, Ansuman; Bhattacharyya, Nitai P

    2011-09-26

    HIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator. We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream

  13. Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

    PubMed

    Ouban, Abderrahman; Ahmed, Atif

    2015-07-01

    Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

  14. An alternatively spliced CXCL16 isoform expressed by dendritic cells is a secreted chemoattractant for CXCR6+ cells.

    PubMed

    van der Voort, Robbert; Verweij, Viviènne; de Witte, Theo M; Lasonder, Edwin; Adema, Gosse J; Dolstra, Harry

    2010-06-01

    DC are professional APCs that initiate and regulate adaptive immune responses by interacting with naïve and memory T cells. Chemokines released by DC play an essential role in T cell recruitment and in the maintenance of antigen-specific T cell-DC conjugates. Here, we characterized the expression of the T cell-attracting chemokine CXCL16 by murine DC. We demonstrate that through alternative RNA splicing, DC not only express the previously characterized transmembrane CXCL16 isoform, which can be cleaved from the cell surface, but also a novel isoform lacking the transmembrane and cytoplasmic domains. Transfection of HEK293 cells shows that this novel isoform, termed CXCL16v, is not expressed on the cell membrane but is secreted as a protein of approximately 10 kDa. Quantitative PCR demonstrates that CXCL16v is broadly expressed in lymphoid and nonlymphoid tissues resembling the tissue distribution of DC. Indeed, CXCL16v mRNA is expressed significantly by spleen DC and BM-DC. Moreover, we show that mature DC have increased CXCL16v mRNA levels and express transmembrane and soluble CXCL16 proteins. Finally, we show that CXCL16v specifically attracts cells expressing the chemokine receptor CXCR6. Our data demonstrate that mature DC express secreted, transmembrane, and cleaved CXCL16 isoforms to recruit and communicate efficiently with CXCR6(+) lymphoid cells.

  15. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    PubMed

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  16. Expression of AID, P53, and Mlh1 proteins in endoscopically resected differentiated-type early gastric cancer

    PubMed Central

    Takeda, Yohei; Yashima, Kazuo; Hayashi, Akihiro; Sasaki, Shuji; Kawaguchi, Koichiro; Harada, Kenichi; Murawaki, Yoshikazu; Ito, Hisao

    2012-01-01

    AIM: To analyze the expression of the tumor-related proteins in differentiated-type early gastric carcinoma (DEGC) samples. METHODS: Tumor specimens were obtained from 102 patients (75 males and 27 females) who had received an endoscopic tumor resection at Tottori University Hospital between 2007 and 2009. Ninety-one cancer samples corresponded to noninvasive or intramucosal carcinoma according to the Vienna classification system, and 11 samples were submucosal invasive carcinomas. All of the EGCs were histologically differentiated carcinomas. All patients were classified as having Helicobacter pylori (H. pylori) infections by endoscopic atrophic changes or by testing seropositive for H. pylori IgG. All of the samples were histopathologically classified as either tubular or papillary adenocarcinoma according to their structure. The immunohistochemical staining was performed in a blinded manner with respect to the clinical information. Two independent observers evaluated protein expression. All data were statistically analyzed then. RESULTS: The rates of aberrant activation-induced cytidine deaminase (AID) expression and P53 overexpression were both 34.3% in DEGCs. The expression of Mlh1 was lost in 18.6% of DEGCs. Aberrant AID expression was not significantly associated with P53 overexpression in DEGCs. However, AID expression was associated with the severity of mononuclear cell activity in the non-cancerous mucosa adjacent to the tumor (P = 0.064). The rate of P53 expression was significantly greater in flat or depressed tumors than in elevated tumors. The frequency of Mlh1 loss was significantly increased in distal tumors, elevated gross-type tumors, papillary histological-type tumors, and tumors with a severe degree of endoscopic atrophic gastritis (P < 0.05). CONCLUSION: Aberrant AID expression, P53 overexpression, and the loss of Mlh1 were all associated with clinicopathological features and gastric mucosal alterations in DEGCs. The aberrant expression of AID

  17. Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells.

    PubMed

    Riddick, C A; Ring, W L; Baker, J R; Hodulik, C R; Bigby, T D

    1997-05-15

    The aim of this study was to assess the effect of dexamethasone on 5-lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP-1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium-ionophore-stimulated 5-lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and its activating protein (5-LAP) was also assessed. Dexamethasone increased the stimulated release of 5-lipoxygenase products from both monocytes and THP-1 cells in a dose-dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4-hydrolase-immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5-lipoxygenase pathway expression in both monocytes and in THP-1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP. These results suggest a role for glucocorticoids in the regulation of 5-lipoxygenase pathway expression in mononuclear phagocytes.

  18. Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

    PubMed

    Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

    2015-05-01

    Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.

  19. Evaluation of correlation between CT image features and ERCC1 protein expression in assessing lung cancer prognosis

    NASA Astrophysics Data System (ADS)

    Tan, Maxine; Emaminejad, Nastaran; Qian, Wei; Sun, Shenshen; Kang, Yan; Guan, Yubao; Lure, Fleming; Zheng, Bin

    2014-03-01

    Stage I non-small-cell lung cancers (NSCLC) usually have favorable prognosis. However, high percentage of NSCLC patients have cancer relapse after surgery. Accurately predicting cancer prognosis is important to optimally treat and manage the patients to minimize the risk of cancer relapse. Studies have shown that an excision repair crosscomplementing 1 (ERCC1) gene was a potentially useful genetic biomarker to predict prognosis of NSCLC patients. Meanwhile, studies also found that chronic obstructive pulmonary disease (COPD) was highly associated with lung cancer prognosis. In this study, we investigated and evaluated the correlations between COPD image features and ERCC1 gene expression. A database involving 106 NSCLC patients was used. Each patient had a thoracic CT examination and ERCC1 genetic test. We applied a computer-aided detection scheme to segment and quantify COPD image features. A logistic regression method and a multilayer perceptron network were applied to analyze the correlation between the computed COPD image features and ERCC1 protein expression. A multilayer perceptron network (MPN) was also developed to test performance of using COPD-related image features to predict ERCC1 protein expression. A nine feature based logistic regression analysis showed the average COPD feature values in the low and high ERCC1 protein expression groups are significantly different (p < 0.01). Using a five-fold cross validation method, the MPN yielded an area under ROC curve (AUC = 0.669±0.053) in classifying between the low and high ERCC1 expression cases. The study indicates that CT phenotype features are associated with the genetic tests, which may provide supplementary information to help improve accuracy in assessing prognosis of NSCLC patients.

  20. Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

    PubMed

    Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

    2017-10-15

    Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers. IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and

  1. Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rhee, Juong G.; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD; Li, Daqing

    2007-01-01

    Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into anmore » adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.« less

  2. [Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

    PubMed

    Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

    2014-01-01

    To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

  3. NF-κB Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments1

    PubMed Central

    Pulai, Judit I.; Chen, Hong; Im, Hee-Jeong; Kumar, Sanjay; Hanning, Charles; Hegde, Priti S.; Loeser, Richard F.

    2010-01-01

    Fibronectin fragments (FN-f) that bind to the α5β1 integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β). Constitutive and FN-f-inducible expression of GRO-α and GRO-γ were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1β expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-κB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction. PMID:15843581

  4. Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer.

    PubMed

    Jung, Yoon Yang; Kim, Hye Min; Koo, Ja Seung

    2015-01-01

    The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof. Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52). The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054-22.59, p = 0.043). Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.

  5. Cancer testis antigen OY-TES-1: analysis of protein expression in ovarian cancer with tissue microarrays.

    PubMed

    Fan, R; Huang, W; Luo, B; Zhang, Q M; Xiao, S W; Xie, X X

    2015-01-01

    Revised manuscript accepted for publication March 5, Objectives: The purpose of this study was to determine the potential of cancer testis antigen OY-TES-1 as a vaccine for ovarian cancer (OC). A tissue microarray (TMA) containing 107 samples from OC tissues and 48 samples from OC adjacent tissues was analyzed by immunohistochemistry with the OY-TES-1 polyclonal antibody. The correlation between OY-TES-1 and clinic pathological traits of OC was statistically analyzed. The expression of OY-TES-1 protein was found in 81% (87/107) of OC tissues and 56% (27/48) of OC adjacent tissues. The immunostaining intensity of OY-TES-1 in OC tissues was significantly higher than that in OC adjacent tissues tested (p = 0.040). OC adjacent tissues only demonstrated lower immunostaining intensity, whereas some of OC tissues presented higher immunostaining intensity and majority showed the heterogeneity of protein distribution. There was no statistically significant correlation found between OY-TES-1 expression and any other clinicopathological traits such as age, FIGO stage, pathological grade, and histological type. OY-TES-1 was expressed in OC tissues with a high proportion, and some of OC tissues presented OY-TES-1 expression in high level vs OC adjacent tissues. OY-TES-1 could be an attractive target for immunotherapy for OC in the future.

  6. WHIRLIN INCREASES TRPV1 CHANNEL EXPRESSION AND CELLULAR STABILITY

    PubMed Central

    Ciardo, Maria Grazia; Andrés-Bordería, Amparo; Cuesta, Natalia; Valente, Pierluigi; Camprubí-Robles, María; Yang, Jun; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

    2017-01-01

    The expression and function of TRPV1 is influenced by its interaction with cellular proteins. Here, we identify whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin-TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders. PMID:26516054

  7. Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

    PubMed Central

    Staley, Chris A.; Huang, Amy; Nattestad, Maria; Oshiro, Kristin T.; Ray, Laura E.; Mulye, Tejas; Li, Zhiguo Harry; Le, Thu; Stephens, Justin J.; Gomez, Seth R.; Moy, Allison D.; Nguyen, Jackson C.; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

    2012-01-01

    Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging. PMID:22285974

  8. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

    PubMed

    Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

    2018-01-26

    Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

  9. Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

    PubMed

    Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Jooho; Hengstschläger, Markus; Lubec, Gert

    2006-05-15

    Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND. (c) 2006 Wiley-Liss, Inc.

  10. Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor

    PubMed Central

    Saber, Anne T; Jacobsen, Nicklas R; Bornholdt, Jette; Kjær, Sanna L; Dybdahl, Marianne; Risom, Lotte; Loft, Steffen; Vogel, Ulla; Wallin, Håkan

    2006-01-01

    Background Particulate air pollution has been associated with lung and cardiovascular disease, for which lung inflammation may be a driving mechanism. The pro-inflammatory cytokine, tumor necrosis factor (TNF) has been suggested to have a key-role in particle-induced inflammation. We studied the time course of gene expression of inflammatory markers in the lungs of wild type mice and Tnf-/- mice after exposure to diesel exhaust particles (DEPs). Mice were exposed to either a single or multiple doses of DEP by inhalation. We measured the mRNA level of the cytokines Tnf and interleukin-6 (Il-6) and the chemokines, monocyte chemoattractant protein (Mcp-1), macrophage inflammatory protein-2 (Mip-2) and keratinocyte derived chemokine (Kc) in the lung tissue at different time points after exposure. Results Tnf mRNA expression levels increased late after DEP-inhalation, whereas the expression levels of Il-6, Mcp-1 and Kc increased early. The expression of Mip-2 was independent of TNF if the dose was above a certain level. The expression levels of the cytokines Kc, Mcp-1 and Il-6, were increased in the absence of TNF. Conclusion Our data demonstrate that Tnf is not important in early DEP induced inflammation and rather exerts negative influence on Mcp-1 and Kc mRNA levels. This suggests that other signalling pathways are important, a candidate being one involving Mcp-1. PMID:16504008

  11. T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

    PubMed

    Ueno, Aruto; Arakawa, Fumiko; Abe, Hironori; Matsumoto, Hisanobu; Kudo, Toshio; Asano, Ryutaro; Tsumoto, Kohei; Kumagai, Izumi; Kuroki, Motomu; Kuroki, Masahide

    2002-01-01

    The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

  12. Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi

    Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-{alpha} and PPAR{gamma}. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPAR{gamma} antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPAR{gamma} activations. The results indicate that auraptene activates PPAR{gamma} in adipocytes to control adipocytekines such as adiponectin and MCP-1 andmore » suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.« less

  13. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  14. Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

    PubMed Central

    Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

    2013-01-01

    Background Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1. Methods The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins. Results Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification. Conclusions Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance. PMID:24376514

  15. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis.

    PubMed

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  16. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis

    PubMed Central

    Ren, Bin

    2016-01-01

    Protein kinase D 1 (PKD-1) is a signaling kinase important in fundamental cell functions including migration, proliferation, and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease, and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis. PMID:27200349

  17. Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

    PubMed

    Wang, Ming-Dong; Franklin, Vivian; Sundaram, Meenakshi; Kiss, Robert S; Ho, Kenneth; Gallant, Michel; Marcel, Yves L

    2007-08-03

    Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

  18. Expression of Ki-67 (MIB-1) and GLUT-1 proteins in non-advanced prostatic cancer.

    PubMed

    Luczynska, Elzbieta; Gasinska, Anna; Wilk, Waclaw

    2012-12-01

    The expression of Ki-67 (MIB-1) and glucose transporter-1 (GLUT-1) were evaluated in patients with clinically localized prostate cancer (PC) who had undergone radical prostatectomy with curative intent. 140 low advanced PC specimens were studied. Protein expression was assessed immunohistochemically on tumour sections and expressed as a labelling index, i.e. the percentage of positively stained cells. In the case of Ki-67 nuclear staining and in the case of GLUT-1 membrane and cytoplasmic staining was considered as positive. The patients' mean age was 62.9 ±6.2 years. There were 13 (9.3%) at pTNM stage 1, 78 (55.7%) at stage 2, 40 (28.6%) at stage 3 and 9 (6.4%) at stage 4, respectively. 75 (53.6%) tumours were well differentiated (Gleason score ≤6), 52 (37.1%) moderately differentiated (Gleason score of 7) and 13 (9.3%) poorly differentiated (Gleason score 8-10). The mean pre-operative serum PSA was 9.9 ± SE 0.5 ng/ml, and the mean LI was equal to 8.1 ±0.6% and 29.7 ±2.0%, for MIB-1 and GLUT-1, respectively. Increase of pathological tumor volume and tumor grade was associated with statistically significant growth of PSA (p < 0.011) and MIB-1LI (p < 0.003), however, for GLUT-1 LI the relation was not significant. Ki-67 expression was correlated with PSA levels (p = 0.013) and GLUT-1 scores (p = 0.04). In PC, an increase in the proliferation rate (higher MIB-1LI) in higher pTNM stages and tumour grades may point to Ki-67 as a good marker of biological aggressiveness useful in selecting patients for more aggressive treatment. A correlation between proliferation and GLUT-1 score may be the evidence of active glycolytic metabolism in hypoxic regions.

  19. G protein, phosphorylated-GATA4 and VEGF expression in the hearts of transgenic mice overexpressing β1- and β2-adrenergic receptors

    PubMed Central

    Tae, Hyun-Jin; Petrashevskaya, Natalia; Kim, In Hye; Park, Joon Ha; Lee, Jae-Chul; Won, Moo-Ho; Kim, Yang Hee; Ahn, Ji Hyeon; Park, Jinseu; Choi, Soo Young; Jeon, Yong Hwan

    2017-01-01

    β1- and β2-adrenergic receptors (ARs) regulate cardiac contractility, calcium handling and protein phosphorylation. The present study aimed to examine the expression levels of vascular endothelial growth factor A (VEGF-A) and several G proteins, and the phosphorylation of transcription factor GATA binding protein 4 (GATA4), by western blot analysis, using isolated hearts from 6 month-old transgenic (TG) mice that overexpress β1AR or β2AR. Cardiac contractility/relaxation and heart rate was increased in both β1AR TG and β2AR TG mouse hearts compared with wild type; however, no significant differences were observed between the β1- and β2AR TG mouse hearts. Protein expression levels of inhibitory guanine nucleotide-binding protein (Gi) 2, Gi3 and G-protein-coupled receptor kinase 2 were upregulated in both TG mice, although the upregulation of Gi2 was more prominent in the β2AR TG mice. VEGF-A expression levels were also increased in both TG mice, and were highest in the β1AR TG mice. In addition, the levels of phosphorylated-GATA4 expression were increased in β1- and β2AR TG mice. In conclusion, the present study demonstrated that cardiac contractility/relaxation and heart rate is increased in β1AR TG and β2AR TG mice, and indicated that this increase may be related to the overexpression of G proteins and G-protein-associated proteins. PMID:28487987

  20. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis

    PubMed Central

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-01-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast. PMID:26348709

  1. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

    PubMed

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-09-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

  2. The AICD interacting protein DAB1 is up-regulated in Alzheimer frontal cortex brain samples and causes deregulation of proteins involved in gene expression changes.

    PubMed

    Müller, T; Loosse, C; Schrötter, A; Schnabel, A; Helling, S; Egensperger, R; Marcus, K

    2011-08-01

    AICD is the intracellular subdomain of the amyloid precursor protein thought to play a pivotal role as a potential transcription factor that might be of relevance for the pathophysiology of Alzheimer's disease. For its signal transduction potential AICD requires interacting proteins like FE65 and TIP60. However, many other proteins were described being able to bind to AICD. Here, we studied mRNA levels of AICD interacting proteins and found one of them (DAB1) strongly up-regulated in human post-mortem frontal cortex brain samples of AD patients. Subsequent cell culture experiments revealed that elevated DAB1 level results in the deregulation of the cellular proteome. We found the proliferation associated protein 2G4 as well as the guanine monophosphate synthetase (GMPS) significantly up-regulated in DAB1 over-expressing cells. Both proteins can be involved in cellular transcription processes supporting the hypothesis that DAB1 acts via modification of the AICD-dependent transcriptionally active complex. Of note, expression of the three components of the putative transcription complex (AICD, FE65, and TIP60 (AFT)) also revealed deregulation of the GMPS protein in an opposite fashion. Our results point to a putative relevance of AICD-dependent mechanisms in AD, caused by protein abundance changes of AICD interacting proteins, as shown for DAB1 in this work.

  3. Expression of Y-box-binding protein YB-1 allows stratification into long- and short-term survivors of head and neck cancer patients.

    PubMed

    Kolk, A; Jubitz, N; Mengele, K; Mantwill, K; Bissinger, O; Schmitt, M; Kremer, M; Holm, P S

    2011-12-06

    Histology-based classifications and clinical parameters of head and neck squamous cell carcinoma (HNSCC) are limited in their clinical capacity to provide information on prognosis and treatment choice of HNSCC. The primary aim of this study was to analyse Y-box-binding protein-1 (YB-1) protein expression in different grading groups of HNSCC patients, and to correlate these findings with the disease-specific survival (DSS). We investigated the expression and cellular localisation of the oncogenic transcription/translation factor YB-1 by immunohistochemistry on tissue micro arrays in a total of 365 HNSCC specimens and correlated expression data with clinico-pathological parameters including DSS. Compared with control tissue from healthy individuals, a significantly (P<0.01) increased YB-1 protein expression was observed in high-grade HNSCC patients. By univariate survival data analysis, HNSCC patients with elevated YB-1 protein expression had a significantly (P<0.01) decreased DSS. By multivariate Cox regression analysis, high YB-1 expression and nuclear localisation retained its significance as a statistically independent (P<0.002) prognostic marker for DSS. Within grade 2 group of HNSCC patients, a subgroup defined by high nuclear and cytoplasmic YB-1 levels (co-expression pattern) in the cells of the tumour invasion front had a significantly poorer 5-year DSS rate of only 38% compared with overall 55% for grade 2 patients. Vice versa, the DSS rate was markedly increased to 74% for grade 2 cancer patients with low YB-1 protein expression at the same localisation. Our findings point to the fact that YB-1 expression in combination with histological classification in a double stratification strategy is superior to classical grading in the prediction of tumour progression in HNSCC.

  4. Expression of Y-box-binding protein YB-1 allows stratification into long- and short-term survivors of head and neck cancer patients

    PubMed Central

    Kolk, A; Jubitz, N; Mengele, K; Mantwill, K; Bissinger, O; Schmitt, M; Kremer, M; Holm, P S

    2011-01-01

    Background: Histology-based classifications and clinical parameters of head and neck squamous cell carcinoma (HNSCC) are limited in their clinical capacity to provide information on prognosis and treatment choice of HNSCC. The primary aim of this study was to analyse Y-box-binding protein-1 (YB-1) protein expression in different grading groups of HNSCC patients, and to correlate these findings with the disease-specific survival (DSS). Methods: We investigated the expression and cellular localisation of the oncogenic transcription/translation factor YB-1 by immunohistochemistry on tissue micro arrays in a total of 365 HNSCC specimens and correlated expression data with clinico-pathological parameters including DSS. Results: Compared with control tissue from healthy individuals, a significantly (P<0.01) increased YB-1 protein expression was observed in high-grade HNSCC patients. By univariate survival data analysis, HNSCC patients with elevated YB-1 protein expression had a significantly (P<0.01) decreased DSS. By multivariate Cox regression analysis, high YB-1 expression and nuclear localisation retained its significance as a statistically independent (P<0.002) prognostic marker for DSS. Within grade 2 group of HNSCC patients, a subgroup defined by high nuclear and cytoplasmic YB-1 levels (co-expression pattern) in the cells of the tumour invasion front had a significantly poorer 5-year DSS rate of only 38% compared with overall 55% for grade 2 patients. Vice versa, the DSS rate was markedly increased to 74% for grade 2 cancer patients with low YB-1 protein expression at the same localisation. Conclusion: Our findings point to the fact that YB-1 expression in combination with histological classification in a double stratification strategy is superior to classical grading in the prediction of tumour progression in HNSCC. PMID:22095225

  5. EFFECT OF THAI SARAPHI FLOWER EXTRACTS ON WT1 AND BCR/ABL PROTEIN EXPRESSION IN LEUKEMIC CELL LINES.

    PubMed

    Sangkaruk, Rungkarn; Rungrojsakul, Methee; Tima, Singkome; Anuchapreeda, Songyot

    2017-01-01

    Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC 50 values of 2.6 and 77.6 μg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WTlprotein expressions by 75 and 49.5%, respectively when compared to vehicle control. The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.

  6. Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

    PubMed Central

    Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

    2014-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

  7. Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

    PubMed

    Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

    2001-04-01

    Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

  8. L-Arogenate is a chemoattractant which can be utilized as the sole source of carbon and nitrogen by Pseudomonas aeruginosa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fischer, R.S.; Song, Jian; Gu, Wei

    L-Arogenate is a commonplace amino acid in nature in consideration of its role as a ubiquitous precursor of L-phenylalanine and/or L-tyrosine. However, the questions of whether it serves as a chemoattractant molecule and whether it can serve as a substrate for catabolism have never been studied. We found that Pseudomonas aeruginosa recognizes L-arogenate as a chemoattractant molecule which can be utilized as a source of both carbon and nitrogen. Mutants lacking expression of either cyclohexadienyl dehydratase or phenylalanine hydroxylase exhibited highly reduced growth rates when utilizing L-arogenate as a nitrogen source. Utilization of L-arogenate as a source of either carbonmore » or nitrogen was dependent upon {sub S}{sup 54}, as revealed by the use of an rpoN null mutant. The evidence suggests that catabolism of L-arogenate proceeds via alternative pathways which converge at 4-hydroxyphenylpyruvate. In one pathway, prephenate formed in the periplasm by deamination of L-arogenate is converted to 4-hydroxyphenylpyruvate by cyclohexadienyl dehydrogenase. The second route depends upon the sequential action of periplasmic cyclohexadienyl dehydratase, phenylalanine hydroxylase, and aromatic aminotransferase. 32 refs., 5 figs., 4 tabs.« less

  9. Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

    PubMed

    Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

    2009-09-01

    Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

  10. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

    PubMed

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

  11. Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity

    PubMed Central

    Verdeguer, Francisco; Soustek, Meghan S.; Hatting, Maximilian; Blättler, Sharon M.; McDonald, Devin; Barrow, Joeva J.

    2015-01-01

    Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight. PMID:26503783

  12. Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation

    PubMed Central

    2011-01-01

    Background HIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator. Results We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in

  13. Thyroid hormone and COUP-TF1 regulate kallikrein-binding protein (KBP) gene expression.

    PubMed

    Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen; Brent, Gregory A

    2011-03-01

    Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

  14. Thyroid Hormone and COUP-TF1 Regulate Kallikrein-Binding Protein (KBP) Gene Expression

    PubMed Central

    Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen

    2011-01-01

    Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T3 and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5′ flanking region (−53 to −29) and nTRE2, located in the first intron (104–132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T3. COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T3 repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T3. Nuclear corepressor knockdown resulted in loss of T3 repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T3 induction of positive thyroid hormone response elements, reverses T3 repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T3 and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T3. PMID:21266512

  15. Modelling of Dictyostelium discoideum movement in a linear gradient of chemoattractant.

    PubMed

    Eidi, Zahra; Mohammad-Rafiee, Farshid; Khorrami, Mohammad; Gholami, Azam

    2017-11-15

    Chemotaxis is a ubiquitous biological phenomenon in which cells detect a spatial gradient of chemoattractant, and then move towards the source. Here we present a position-dependent advection-diffusion model that quantitatively describes the statistical features of the chemotactic motion of the social amoeba Dictyostelium discoideum in a linear gradient of cAMP (cyclic adenosine monophosphate). We fit the model to experimental trajectories that are recorded in a microfluidic setup with stationary cAMP gradients and extract the diffusion and drift coefficients in the gradient direction. Our analysis shows that for the majority of gradients, both coefficients decrease over time and become negative as the cells crawl up the gradient. The extracted model parameters also show that besides the expected drift in the direction of the chemoattractant gradient, we observe a nonlinear dependency of the corresponding variance on time, which can be explained by the model. Furthermore, the results of the model show that the non-linear term in the mean squared displacement of the cell trajectories can dominate the linear term on large time scales.

  16. Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1

    PubMed Central

    Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

    2017-01-01

    As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated. PMID:27451975

  17. Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

    PubMed

    Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

    2017-02-01

    As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

  18. A New Protein Phosphatase 2C (FsPP2C1) Induced by Abscisic Acid Is Specifically Expressed in Dormant Beechnut Seeds1

    PubMed Central

    Lorenzo, Oscar; Rodríguez, Dolores; Nicolás, Gregorio; Rodríguez, Pedro L.; Nicolás, Carlos

    2001-01-01

    An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein phosphatase 2C (PP2C) was obtained by means of differential reverse transcriptase-polymerase chain reaction approach. The full-length clone was isolated from a cDNA library constructed using mRNA from ABA-treated beechnut (Fagus sylvatica) seeds. This clone presents all the features of plant type PP2C and exhibits homology to members of this family such as AthPP2CA (61%), ABI1 (48%), or ABI2 (47%), therefore it was named FsPP2C1. The expression of FsPP2C1 is detected in dormant seeds and increases after ABA treatment, when seeds are maintained dormant, but it decreases and tends to disappear when dormancy is being released by stratification or under gibberellic acid treatment. Moreover, drought stress seems to have no effect on FsPP2C1 transcript accumulation. The FsPP2C1 transcript expression is tissue specific and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the corresponding protein could be related to ABA-induced seed dormancy. By expressing FsPP2C1 in Escherichia coli as a histidine tag fusion protein, we have obtained direct biochemical evidence supporting Mg2+-dependent phosphatase activity of this protein. PMID:11299374

  19. A Role for the Nucleosome Assembly Proteins TAF-Iβ and NAP1 in the Activation of BZLF1 Expression and Epstein-Barr Virus Reactivation

    PubMed Central

    Frappier, Lori

    2013-01-01

    The reactivation of Epstein-Barr virus (EBV) from latent to lytic infection begins with the expression of the viral BZLF1 gene, leading to a subsequent cascade of viral gene expression and amplification of the EBV genome. Using RNA interference, we show that nucleosome assembly proteins NAP1 and TAF-I positively contribute to EBV reactivation in epithelial cells through the induction of BZLF1 expression. In addition, overexpression of NAP1 or the β isoform of TAF-I (TAF-Iβ) in AGS cells latently infected with EBV was sufficient to induce BZLF1 expression. Chromatin immunoprecipitation experiments performed in AGS-EBV cells showed that TAF-I associated with the BZLF1 promoter upon lytic induction and affected local histone modifications by increasing H3K4 dimethylation and H4K8 acetylation. MLL1, the host protein known to dimethylate H3K4, was found to associate with the BZLF1 promoter upon lytic induction in a TAF-I-dependent manner, and MLL1 depletion decreased BZLF1 expression, confirming its contribution to lytic reactivation. The results indicate that TAF-Iβ promotes BZLF1 expression and subsequent lytic infection by affecting chromatin at the BZLF1 promoter. PMID:23691099

  20. A role for the nucleosome assembly proteins TAF-Iβ and NAP1 in the activation of BZLF1 expression and Epstein-Barr virus reactivation.

    PubMed

    Mansouri, Sheila; Wang, Shan; Frappier, Lori

    2013-01-01

    The reactivation of Epstein-Barr virus (EBV) from latent to lytic infection begins with the expression of the viral BZLF1 gene, leading to a subsequent cascade of viral gene expression and amplification of the EBV genome. Using RNA interference, we show that nucleosome assembly proteins NAP1 and TAF-I positively contribute to EBV reactivation in epithelial cells through the induction of BZLF1 expression. In addition, overexpression of NAP1 or the β isoform of TAF-I (TAF-Iβ) in AGS cells latently infected with EBV was sufficient to induce BZLF1 expression. Chromatin immunoprecipitation experiments performed in AGS-EBV cells showed that TAF-I associated with the BZLF1 promoter upon lytic induction and affected local histone modifications by increasing H3K4 dimethylation and H4K8 acetylation. MLL1, the host protein known to dimethylate H3K4, was found to associate with the BZLF1 promoter upon lytic induction in a TAF-I-dependent manner, and MLL1 depletion decreased BZLF1 expression, confirming its contribution to lytic reactivation. The results indicate that TAF-Iβ promotes BZLF1 expression and subsequent lytic infection by affecting chromatin at the BZLF1 promoter.

  1. The influence of monovalent cations on trimeric G protein G(i)1α activity in HEK293 cells stably expressing DOR-G(i)1α (Cys(351)-Ile(351)) fusion protein.

    PubMed

    Vošahlíková, M; Svoboda, P

    2011-01-01

    The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved.

  2. Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.

    PubMed

    Gilbert, T L; Bennett, T A; Maestas, D C; Cimino, D F; Prossnitz, E R

    2001-03-27

    After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.

  3. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  4. Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression

    PubMed Central

    Michael, Bindhu; Nair, Amithraj; Lairmore, Michael D.

    2010-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T- lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 “accessory” proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for

  5. PTEN Loss Increases PD-L1 Protein Expression and Affects the Correlation between PD-L1 Expression and Clinical Parameters in Colorectal Cancer

    PubMed Central

    Lu, Biyan; Wang, Chenliang; Zhang, Junxiao; Huang, Lanlan; Wang, Xiaoyan; Timmons, Christine L.; Hu, Jun; Liu, Bindong; Wu, Xiaojian; Wang, Lei; Wang, Jianping; Liu, Huanliang

    2013-01-01

    Background Programmed death ligand-1 (PD-L1) has been identified as a factor associated with poor prognosis in a range of cancers, and was reported to be mainly induced by PTEN loss in gliomas. However, the clinical effect of PD-L1 and its regulation by PTEN has not yet been determined in colorectal cancer (CRC). In the present study, we verified the regulation of PTEN on PD-L1 and further determined the effect of PTEN on the correlation between PD-L1 expression and clinical parameters in CRC. Methods/Results RNA interference approach was used to down-regulate PTEN expression in SW480, SW620 and HCT116 cells. It was showed that PD-L1 protein, but not mRNA, was significantly increased in cells transfected with siRNA PTEN compared with the negative control. Moreover, the capacity of PTEN to regulate PD-L1 expression was not obviously affected by IFN-γ, the main inducer of PD-L1. Tissue microarray immunohistochemistry was used to detect PD-L1 and PTEN in 404 CRC patient samples. Overexpression of PD-L1 was significantly correlated with distant metastasis (P<0.001), TNM stage (P<0.01), metastatic progression (P<0.01) and PTEN expression (P<0.001). Univariate analysis revealed that patients with high PD-L1 expression had a poor overall survival (P<0.001). However, multivariate analysis did not support PD-L1 as an independent prognostic factor (P = 0.548). Univariate (P<0.001) and multivariate survival (P<0.001) analysis of 310 located CRC patients revealed that high level of PD-L1 expression was associated with increased risks of metastatic progression. Furthermore, the clinical effect of PD-L1 on CRC was not statistically significant in a subset of 39 patients with no PTEN expression (distant metastasis: P = 0.102; TNM stage: P = 0.634, overall survival: P = 0.482). Conclusions PD-L1 can be used to identify CRC patients with high risk of metastasis and poor prognosis. This clinical manifestation may be partly associated with PTEN expression. PMID

  6. PTEN loss increases PD-L1 protein expression and affects the correlation between PD-L1 expression and clinical parameters in colorectal cancer.

    PubMed

    Song, Minmin; Chen, Defeng; Lu, Biyan; Wang, Chenliang; Zhang, Junxiao; Huang, Lanlan; Wang, Xiaoyan; Timmons, Christine L; Hu, Jun; Liu, Bindong; Wu, Xiaojian; Wang, Lei; Wang, Jianping; Liu, Huanliang

    2013-01-01

    Programmed death ligand-1 (PD-L1) has been identified as a factor associated with poor prognosis in a range of cancers, and was reported to be mainly induced by PTEN loss in gliomas. However, the clinical effect of PD-L1 and its regulation by PTEN has not yet been determined in colorectal cancer (CRC). In the present study, we verified the regulation of PTEN on PD-L1 and further determined the effect of PTEN on the correlation between PD-L1 expression and clinical parameters in CRC. RNA interference approach was used to down-regulate PTEN expression in SW480, SW620 and HCT116 cells. It was showed that PD-L1 protein, but not mRNA, was significantly increased in cells transfected with siRNA PTEN compared with the negative control. Moreover, the capacity of PTEN to regulate PD-L1 expression was not obviously affected by IFN-γ, the main inducer of PD-L1. Tissue microarray immunohistochemistry was used to detect PD-L1 and PTEN in 404 CRC patient samples. Overexpression of PD-L1 was significantly correlated with distant metastasis (P<0.001), TNM stage (P<0.01), metastatic progression (P<0.01) and PTEN expression (P<0.001). Univariate analysis revealed that patients with high PD-L1 expression had a poor overall survival (P<0.001). However, multivariate analysis did not support PD-L1 as an independent prognostic factor (P = 0.548). Univariate (P<0.001) and multivariate survival (P<0.001) analysis of 310 located CRC patients revealed that high level of PD-L1 expression was associated with increased risks of metastatic progression. Furthermore, the clinical effect of PD-L1 on CRC was not statistically significant in a subset of 39 patients with no PTEN expression (distant metastasis: P = 0.102; TNM stage: P = 0.634, overall survival: P = 0.482). PD-L1 can be used to identify CRC patients with high risk of metastasis and poor prognosis. This clinical manifestation may be partly associated with PTEN expression.

  7. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

    PubMed Central

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285

  8. Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial-mesenchymal transition.

    PubMed

    Kolesnikoff, Natasha; Attema, Joanne L; Roslan, Suraya; Bert, Andrew G; Schwarz, Quenten P; Gregory, Philip A; Goodall, Gregory J

    2014-04-18

    Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state. However, it remains unclear what factors drive and maintain epithelial-specific expression of miR-200 in the absence of EMT-inducing factors. Here, we show that the transcription factor Specificity Protein 1 (Sp1) binds to the miR-200b∼200a∼429 proximal promoter and activates miR-200 expression in epithelial cells. In mesenchymal cells, Sp1 expression is maintained, but its ability to activate the miR-200 promoter is perturbed by ZEB-mediated repression. Reduction of Sp1 expression caused changes in EMT-associated markers in epithelial cells. Furthermore, we observed co-expression of Sp1 and miR-200 during mouse embryonic development wherein miR-200 expression was only lost in regions with high ZEB expression. Together, these findings indicate that miR-200 family members require Sp1 to drive basal expression and to maintain an epithelial state.

  9. The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression

    PubMed Central

    Balda, Maria S.; Matter, Karl

    2000-01-01

    Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation. PMID:10790369

  10. Down-regulation of monocarboxylate transporter 1 (MCT1) gene expression in the colon of piglets is linked to bacterial protein fermentation and pro-inflammatory cytokine-mediated signalling.

    PubMed

    Villodre Tudela, Carmen; Boudry, Christelle; Stumpff, Friederike; Aschenbach, Jörg R; Vahjen, Wilfried; Zentek, Jürgen; Pieper, Robert

    2015-02-28

    The present study investigated the influence of bacterial metabolites on monocarboxylate transporter 1 (MCT1) expression in pigs using in vivo, ex vivo and in vitro approaches. Piglets (n 24) were fed high-protein (26 %) or low-protein (18 %) diets with or without fermentable carbohydrates. Colonic digesta samples were analysed for a broad range of bacterial metabolites. The expression of MCT1, TNF-α, interferon γ (IFN-γ) and IL-8 was determined in colonic tissue. The expression of MCT1 was lower and of TNF-α and IL-8 was higher with high-protein diets (P< 0·05). MCT1 expression was positively correlated with l-lactate, whereas negatively correlated with NH₃ and putrescine (P< 0·05). The expression of IL-8 and TNF-α was negatively correlated with l-lactate and positively correlated with NH₃ and putrescine, whereas the expression of IFN-γ was positively correlated with histamine and 4-ethylphenol (P< 0·05). Subsequently, porcine colonic tissue and Caco-2 cells were incubated with Na-butyrate, NH₄Cl or TNF-α as selected bacterial metabolites or mediators of inflammation. Colonic MCT1 expression was higher after incubation with Na-butyrate (P< 0·05) and lower after incubation with NH₄Cl or TNF-α (P< 0·05). Incubation of Caco-2 cells with increasing concentrations of these metabolites confirmed the up-regulation of MCT1 expression by Na-butyrate (linear, P< 0·05) and down-regulation by TNF-α and NH₄Cl (linear, P< 0·05). The high-protein diet decreased the expression of MCT1 in the colon of pigs, which appears to be linked to NH₃- and TNF-α-mediated signalling.

  11. Y Box-Binding Protein 1 Promotes Epithelial-Mesenchymal Transition, Invasion, and Metastasis of Cervical Cancer via Enhancing the Expressions of Snail.

    PubMed

    Pang, Tianyun; Li, Min; Zhang, Ye; Yong, Weiwei; Kang, Haixian; Yao, Yunhong; Hu, Xinrong

    2017-10-01

    Y box-binding protein 1 (YB-1) is a potent oncogenic protein. How it regulates Snail in most tumors including cervical cancer is unknown. This article is to study if YB-1 plays a role in cervical cancer via regulating the expression of Snail. Immunohistochemical staining of YB-1, Snail, and E-cadherin (E-cad) was performed on tissue specimens including 35 cases of chronic cervicitis (as a control), 35 cases of cervical intraepithelial neoplasm (CIN) I, 35 cases of CIN II/III, 28 cases of unmetastatic cervical squamous cell carcinoma, and 19 cases of metastatic cervical squamous cell carcinoma. RNA interference technique was used to knock down YB-1, E6, and Snail genes. Quantitative polymerase chain reaction, western blot, and transwell experiment were used to detect RNA, protein, and cell invasion of cervical cancer cell lines Hela and C33A, respectively. First, YB-1 knockdown significantly reduced messenger RNA (mRNA) and protein levels of Snail, followed by the increased mRNA and protein levels of E-cad and the decreased invasive ability in both Hela (human papillomavirus [HPV] 18+) and C33A (HPV-) cell lines. Second, YB-1 and Snail protein were correlatively expressed in the group order of metastatic cervical squamous cell carcinoma > unmetastatic cervical squamous cell carcinoma > CINs > cervicitis, with the inverse expression mode of E-cad in the group order, P value less than 0.01, between any 2 groups. Finally, HPV18 E6 knockdown reduced the mRNA and protein levels of YB-1 and Snail in Hela cells. The results firstly reported that YB-1 whose mRNA expression is regulated by HPV18 E6 promotes epithelial-mesenchymal transition and progression of cervical cancer via enhancing the expressions of Snail, which indicated that YB-1/Snail/epithelial-mesenchymal transition axis could have a potential use in the diagnosis and therapy of cervical cancer metastasis as a cancer marker and molecular target.

  12. A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells.

    PubMed

    Choi, Yoon Kyung

    2017-12-01

    Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF 165 ) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF 165 did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF 165 -mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF 165 -induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2-PKCζ axis in HRMECs.

  13. Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

    PubMed

    Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

    2017-04-28

    Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

  14. Cdx1 and cdx2 expression during intestinal development.

    PubMed

    Silberg, D G; Swain, G P; Suh, E R; Traber, P G

    2000-10-01

    The intestine-specific transcription factors Cdx1 and Cdx2 are candidate genes for directing intestinal development, differentiation, and maintenance of the intestinal phenotype. This study focused on the complex patterns of expression of Cdx1 and Cdx2 during mouse gastrointestinal development. Embryonic and postnatal mouse tissues were analyzed by immunohistochemistry to determine protein expression of Cdx1 and Cdx2 in the developing intestinal tract. Cdx2 protein expression was observed at 9. 5 postcoitum (pc), whereas weak expression of Cdx1 protein was first seen at 12.5 pc in the distal developing intestine (hindgut). Expression of Cdx1 increased from 13.5 to 14.5 pc during the endoderm/epithelial transition with predominately distal expression. In contrast to Cdx1, there was intense expression of Cdx2 in all but the distal portions of the developing intestine. Cdx2 expression remained low in the distal colon throughout postnatal development. A gradient of expression formed in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. Direct comparison of the patterns of Cdx1 and Cdx2 protein expression during development as performed in this study provides new insights into their potential functional roles. The relative expression of Cdx1 to Cdx2 protein may be important in the anterior to posterior patterning of the intestinal epithelium and in defining patterns of proliferation and differentiation along the crypt-villus axis.

  15. Changes in protein and gene expression of angiotensin II receptors (AT1 and AT2) in aorta of diabetic and hypertensive rats.

    PubMed

    Romero-Nava, R; Rodriguez, J E; Reséndiz-Albor, A A; Sánchez-Muñoz, F; Ruiz-Hernandéz, A; Huang, F; Hong, E; Villafaña, S

    2016-01-01

    Diabetes and hypertension have been associated with cardiovascular diseases and stroke. Some reports have related the coexistence of hypertension and diabetes with increase in the risk of developing vascular complications. Recently some studies have shown results suggesting that in the early stages of diabetes and hypertension exist a reduced functional response to vasopressor agents like angiotensin II (Ang II), which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of AT1 and AT2 receptors in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes, the protein expression was obtained by western blot and the mRNA expression by RT-PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1 receptors than normotensive rats while the AT2 expression remained unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion, our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT1 and AT2 receptors. However, the overexpression of AT2 could be associated with the reduction in the response to Ang II in the early stage of diabetes.

  16. Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

    PubMed

    Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

    2015-02-01

    Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

    PubMed Central

    Ahern, Chris A; Vallejo, Paola; Mortenson, Lindsay; Coronado, Roberto

    2001-01-01

    Background The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. Results Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S) expressed the N-terminal half of α1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. Discussion A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel. PMID:11806762

  18. Endothelial protein C receptor is overexpressed in rheumatoid arthritic (RA) synovium and mediates the anti‐inflammatory effects of activated protein C in RA monocytes

    PubMed Central

    Xue, Meilang; March, Lyn; Sambrook, Philip N; Fukudome, Kenji; Jackson, Christopher J

    2007-01-01

    Objectives (1) To investigate whether inflammatory synovial tissues from patients with rheumatoid arthritis (RA) express endothelial protein C receptor (EPCR) and (2) to determine the major cell type(s) that EPCR is associated with and whether EPCR functions to mediate the effects of activated protein C (APC) on these cells. Methods EPCR, CD68 and PC/APC in synovial tissues were detected by immunostaining and in situ PCR. Monocytes were isolated from peripheral blood of patients with RA and treated with APC, lipopolysaccharide (LPS), and/or EPCR blocking antibody RCR252. Cells and supernatants were collected for RT‐PCR, western blotting, enzyme‐linked immuosorbent assay and chemotaxis assay. Results: EPCR was expressed by both OA and RA synovial tissues but was markedly increased in RA synovium. EPCR was colocalised with PC/APC mostly on CD68 positive cells in synovium. In RA monocytes, APC upregulated EPCR expression and reduced monocyte chemoattractant protein‐1‐induced chemotaxis of monocytes by approximately 50%. APC also completely suppressed LPS‐stimulated NF‐κB activation and attenuated TNF‐α protein by more than 40% in RA monocytes. The inhibitory effects of APC were reversed by RCR252, indicating that EPCR is required. Conclusions Our results demonstrate for the first time that EPCR is expressed by synovial tissues, particularly in RA, where it co‐localises with PC/APC on monocytes/macrophages. In addition, APC inhibits the migration and activation of RA monocytes via EPCR. These inhibitory effects on RA monocytes suggest that PC pathway may have a beneficial therapeutic effect in RA. PMID:17491095

  19. Solanum tuberosum ZPR1 encodes a light-regulated nuclear DNA-binding protein adjusting the circadian expression of StBBX24 to light cycle.

    PubMed

    Kiełbowicz-Matuk, Agnieszka; Czarnecka, Jagoda; Banachowicz, Ewa; Rey, Pascal; Rorat, Tadeusz

    2017-03-01

    ZPR1 proteins belong to the C4-type of zinc finger coordinators known in animal cells to interact with other proteins and participate in cell growth and proliferation. In contrast, the current knowledge regarding plant ZPR1 proteins is very scarce. Here, we identify a novel potato nuclear factor belonging to this family and named StZPR1. StZPR1 is specifically expressed in photosynthetic organs during the light period, and the ZPR1 protein is located in the nuclear chromatin fraction. From modelling and experimental analyses, we reveal the StZPR1 ability to bind the circadian DNA cis motif 'CAACAGCATC', named CIRC and present in the promoter of the clock-controlled double B-box StBBX24 gene, the expression of which peaks in the middle of the day. We found that transgenic lines silenced for StZPR1 expression still display a 24 h period for the oscillation of StBBX24 expression but delayed by 4 h towards the night. Importantly, other BBX genes exhibit altered circadian regulation in these lines. Our data demonstrate that StZPR1 allows fitting of the StBBX24 circadian rhythm to the light period and provide evidence that ZPR1 is a novel clock-associated protein in plants necessary for the accurate rhythmic expression of specific circadian-regulated genes. © 2016 John Wiley & Sons Ltd.

  20. Expression of monocarboxylate transporter 1 (MCT1) in the dog intestine.

    PubMed

    Shimoyama, Yumiko; Kirat, Doaa; Akihara, Yuko; Kawasako, Kazufumi; Komine, Misa; Hirayama, Kazuko; Matsuda, Kazuya; Okamoto, Minoru; Iwano, Hidetomo; Kato, Seiyu; Taniyama, Hiroyuki

    2007-06-01

    In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.

  1. Pig has no uncoupling protein 1.

    PubMed

    Hou, Lianjie; Shi, Jia; Cao, Lingbo; Xu, Guli; Hu, Chingyuan; Wang, Chong

    2017-06-10

    Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. Uncoupling protein 1 (UCP1) is responsible for the non-shivering thermogenesis in the BAT. Pig is important economically as a meat-producing livestock. However, whether BAT or more precisely UCP1 protein exists in pig remains a controversy. The objective of this study was to ascertain whether pig has UCP1 protein. In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3' end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. Then we cloned the pig UCP1 gene exons 1 and 2, and expressed the UCP1 protein from the truncated pig gene using E. coli BL21. We used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. We could not detect any UCP1 protein expression in different pig adipose tissues by the specific pig UCP1 antibody, while our antibody can detect the cloned pig UCP1 as well as the mice adipose UCP1 protein. This result shows although exons 1 and 2 of the pig UCP1 gene were transcribed but not translated in the pig adipose tissue. Furthermore, we detected no uncoupled respiration in the isolated pig adipocytes. Thus, these results unequivocally demonstrate that pig has no UCP1 protein. Our results have resolved the controversy of whether pigs have the brown adipose tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1.

    PubMed

    Majumder, Pritha; Chattopadhyay, Biswanath; Mazumder, Arindam; Das, Pradeep; Bhattacharyya, Nitai P

    2006-05-01

    To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

  3. A 52-week safety study in cynomolgus macaques for genetically modified rice expressing Cry1Ab/1Ac protein.

    PubMed

    Mao, Jie; Sun, Xing; Cheng, Jian-Hua; Shi, Yong-Jie; Wang, Xin-Zheng; Qin, Jun-Jie; Sang, Zhi-Hong; He, Kun; Xia, Qing

    2016-09-01

    A 52-week feeding study in cynomolgus macaques was carried out to evaluate the safety of Bt rice Huahui 1 (HH1), a transgenic rice line expressing Cry1Ab/1Ac protein. Monkeys were fed a diet with 20% or 60% HH1 rice, 20% or 60% parental rice (Minghui 63, MH63), normal diet, normal diet spiked with purified recombinant Cry1Ab/1Ac fusion protein or bovine serum albumin (BSA) respectively. During the feeding trail, clinical observations were conducted daily, and multiple parameters, including body weight, body temperature, electrocardiogram, hematology, blood biochemistry, serum metabolome and gut microbiome were examined at regular intervals. Upon sacrifice, the organs were weighted, and the macroscopic, microscopic and electron microscopic examinations were performed. The results show no adverse or toxic effects of Bt rice HH1 or Cry1Ab/1Ac fusion protein on monkeys. Therefore, the present 52-week primate feeding study suggests that the transgenic rice containing Cry 1Ab/1Ac is equivalent to its parental rice line MH63. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Expression of DNA repair proteins MSH2, MLH1 and MGMT in human benign and malignant thyroid lesions: An immunohistochemical study

    PubMed Central

    Giaginis, Constantinos; Michailidi, Christina; Stolakis, Vasileios; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Klijanienko, Jerzy; Delladetsima, Ioanna; Theocharis, Stamatios

    2011-01-01

    Summary Background DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. Material/Methods MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. Results The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p=0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p=0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p=0.014 and p=0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p=0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p=0.006 and p=0.002, respectively). Conclusions Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia. PMID:21358597

  5. [Alteration of mitochondrial distribution and gene expression of fission 1 protein in cortical neurons of rats with chronic fluorosis].

    PubMed

    Lou, Di-dong; Zhang, Kai-lin; Qin, Shuang-li; Liu, Yan-fei; Yu, Yan-ni; Guan, Zhi-zhong

    2012-04-01

    To investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 (Fis1) protein in the cortical neurons of rats with chronic fluorosis. Sixty SD rats were divided into 3 groups (20 each) according to weight hierarchy and fed with different concentrations of fluoride in drinking water (0, 10 and 50 mg/L, respectively) for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COXIV and tubulin-α were captured with fluorescence microscope. Fis1 protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fis1 mRNA was detected with real-time PCR. Varying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COXIV(34.8 ± 4.7 and 39.3 ± 3.0, respectively), and the expression of Fis1 protein (immunohistochemistry: 54.0 ± 3.6 and 51.3 ± 4.1, respectively; Western blot: 2.9 ± 0.4 and 2.6 ± 0.6, respectively) and mRNA (3773 ± 1292 and 1274 ± 162, respectively) was markedly increased as compared with controls (4.4 ± 2.3, 25.2 ± 2.5, 1.8 ± 0.2 and 277 ± 73) over the experimental period of 6 months. Excessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons (i.e., the increase in soma and the decrease in axon), increased expression of Fis1 gene and enhanced mitochondrial fission. The altered mitochondrial distribution may be related to the high expression level of Fis1 and a functional disorder of mitochondria.

  6. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  7. [Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

    PubMed

    Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

    2011-05-01

    To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

  8. [Correlation between EGLN1 gene, protein express in lung tissue of rats and pulmonary artery pressure at different altitude].

    PubMed

    Li, S H; Li, S; Sun, L; Bai, Z Z; Yang, Q Y; Ga, Q; Jin, G E

    2016-08-23

    To investigate the correlation between pulmonary artery pressure (PAP) and the expression level of Egl nine homologue 1 (EGLN1) gene or its protein in lung tissue of rats at different altitudes. Totally 121 male Wistar rats were randomly divided into low altitude group (n=11), moderate altitude group and high altitude group, the rats in moderate altitude and high altitude group were further divided into 1(st) day, 3(rd) days, 7(th) days, 15(th) day and 30(th) day group according to the exposure time to hypoxic environment, each group 11 rats. The low altitude group, the PAP of rats were determined by physiological signal acquisition system, and tissue samples were collected in liquid nitrogen container for storage at an altitude of 498 m area. Moderate altitude group rats were placed in altitude of 2 260 meters of natural environment, 5 high altitude groups rats were placed in the hypobaric hypoxic chamber, simulating altitude of 4 500 meters. The PAP of rats in moderate altitude group and high altitude group were also determined by physiological signal acquisition system, and tissue samples were collected when rats were exposed to hypoxia at 1(st), 3(rd), 7(th), 15(th) and 30(th) day; Western blot was used to determine expression levels of EGLN1 protein, and person correlation analysis was used to analyze whether the protein was related to the formation of pulmonary arterial hypertension (PH) under hypoxia. Real-time quantitive PCR method determined expression levels of EGLN1 mRNA in lung tissues, and the relative expression method was used to analyze PCR data, and finally assess whether the EGLN1 gene was the initial cause of the formation of PH during hypoxia. The mean PAP of rats was (20.0±3.2) mmHg (1 mmHg=0.133 kPa) in low altitude group; in moderate altitude group, mean PAP began to increase slightly when rats were exposed to hypoxia on the 15(th) day and reached at (22.7±4.1) mmHg on hypoxic 30(th) day, but compared with the low altitude group, there was

  9. [Gene copy number, mRNA transcription and protein expression of PD-1 gene in primary hepatocarcinoma patients].

    PubMed

    Fan, Hui-Min; Wu, Ling-Jie; Hu, Feng-Yu; Yang, Zhan

    2012-08-01

    To study the gene copy number, mRNA transcription and protien expression of programmed cell death 1 (PD-1) gene in primary hepatocellular carcinoma (PHC) patients and normal control individuals (NC) who are anti-HBs positive, and to investigate the variations in PD-1 gene copy numbers and its relationship with PHC. Real-time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 24 samples of PHC patients and 26 of NC. Protein expression level of PD-1 on CD8+ T was analyzed by flow cytometry. In terms of number of PD-1 gene copy numbers, the percentage of cases of haploid (single) was 34.62% and 4.17% in PHC group and control group respectively while the percentage of cases of diploid (double) was 61.54% and 95.83% respectively. The difference between the two was statistically significant (chi2 = 7.639, P = 0.006). The rate of cases with double PD-1 gene copy numbers was found to be higher in patients with PHC than in control group. It was also found that the average expression of PD-1 mRNA was 2.35E-03 in control group and 1.23E-03 in PHC group. The expression level was significant lower in PHC group than that in control group when compared by using Mann-whitey technic (U = 153, P = 0.009). Furthermore, the frequency of PD-1 protein expression on CD8+ T cells was 3.72 +/- 0.32 in control group and 16.13 +/- 1.68 in PHC group. The level of PD-1 mRNA expression was higher in PHC and significant differences was shown between two groups (t = -7.073, P = 0.000). Our study suggests that the variation in PD-1 gene copy number may trigger primary hepatocellular carcinoma to HBV carriers. The relationship between the variation of PD-1 gene copy numbers and its association with primary hepatocellular carcinoma is worth further focus.

  10. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  11. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    PubMed

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

  12. Arborvitae (Thuja plicata) essential oil significantly inhibited critical inflammation- and tissue remodeling-related proteins and genes in human dermal fibroblasts.

    PubMed

    Han, Xuesheng; Parker, Tory L

    2017-06-01

    Arborvitae ( Thuja plicata ) essential oil (AEO) is becoming increasingly popular in skincare, although its biological activity in human skin cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to inflammation and tissue remodeling by using a pre-inflamed human dermal fibroblast culture model. AEO significantly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1), intracellular cell adhesion molecule 1 (ICAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell chemoattractant (I-TAC), monokine induced by interferon gamma (MIG), and macrophage colony-stimulating factor (M-CSF). It also showed significant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1 (PAI-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2). The inhibitory effect of AEO on increased production of these protein biomarkers suggests it has anti-inflammatory property. We then studied the effect of AEO on the genome-wide expression of 21,224 genes in the same cell culture. AEO significantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA) showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inflammatory and tissue remodeling processes. The findings of this study provide the first evidence of the biological activity and beneficial action of AEO in human skin cells.

  13. Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species.

    PubMed

    Li, Wentao; Chetelat, Roger T

    2015-04-07

    Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin-proteasome pathway, a mechanism related to that which controls pollen recognition in SI.

  14. Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

    PubMed

    Moghanibashi, Mehdi; Rastgar Jazii, Ferdous; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

    2013-06-01

    Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

  15. Expression of the glutamine metabolism-related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours.

    PubMed

    Ryu, J-E; Park, H-K; Choi, H-J; Lee, H-B; Lee, H-J; Lee, H; Yu, E-S; Son, W-C

    2018-06-01

    Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism-related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex-type tumours displayed significantly higher GLS1 intensity than simple-type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer. © 2017 John Wiley & Sons Ltd.

  16. Marker Protein Expression Combined With Expression Heterogeneity is a Powerful Indicator of Malignancy in Acral Lentiginous Melanomas.

    PubMed

    Cintra Lopes Carapeto, Fernando; Neves Comodo, Andréia; Germano, Andressa; Pereira Guimarães, Daiane; Barcelos, Denise; Fernandes, Mariana; Landman, Gilles

    2017-02-01

    Samples of acral lentiginous melanomas (ALMs) were obtained from the Department of Pathology at Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Demographic, clinical, and follow-up data were obtained from the charts of Hospital São Paulo. From 2 tissue microarrays containing 60 nevi and quadruplicate samples of ≥1.0-mm of 49 ALM, sections were stained to evaluate SCF, KIT, BRAF, CYCLIND1, MYC, and PTEN immunohistochemical protein expression. Nevi and ALM from 2006 to 2010 were reviewed and collected. All specimens were in the vertical growth phase, and histopathological parameters indicated that tumors were at an advanced stage at diagnosis. Average tumor thickness was 6.95 mm, 63% were ulcerated, average mitotic index was 5 mitotic cells per mm, and 43% were at Clark's level V. Compared with nevi, the χ test showed that ALM significantly correlated with SCF protein expression (P = 0.001) and expression heterogeneity (P < 0.000). Similar findings were observed for KIT (P = 0.005, P = 0.003, respectively), MYC (P < 0.000, P < 0.000), and PTEN (P = 0.005, P < 0.000). Malignancy did not correlate with BRAF and CYCLIN D1 expression (P = 0.053 and P = 0.259, respectively), but it did significantly correlate with their heterogeneous expression (P < 0.000, P = 0.024, respectively). Combined protein expression had an odds ratio of greater malignancy when BRAF and MYC were positive and/or heterogeneously expressed (OR of 78 and 95, respectively). We show that marker protein expression, when combined with heterogeneous expression as shown by immunohistochemistry, is a powerful indicator of malignancy in ALMs, especially, when protein pairs are combined.

  17. Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

    PubMed

    Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

    2004-10-27

    Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

  18. Protein-disulfide Isomerase Regulates the Thyroid Hormone Receptor-mediated Gene Expression via Redox Factor-1 through Thiol Reduction-Oxidation*

    PubMed Central

    Hashimoto, Shoko; Imaoka, Susumu

    2013-01-01

    Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3′,5-triiodothyronine (T3)-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T3-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T3-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRβ1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function. PMID:23148211

  19. Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

    PubMed

    Bozdogan, Onder; Yulug, Isik G; Vargel, Ibrahim; Cavusoglu, Tarik; Karabulut, Ayse A; Karahan, Gurbet; Sayar, Nilufer

    2015-08-01

    Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors. © 2014 The International Society of Dermatology.

  20. Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

    PubMed

    Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

    2015-08-01

    To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its

  1. Osthole protects against inflammation in a rat model of chronic kidney failure via suppression of nuclear factor-κB, transforming growth factor-β1 and activation of phosphoinositide 3-kinase/protein kinase B/nuclear factor (erythroid-derived 2)-like 2 signaling.

    PubMed

    Huang, Tao; Dong, Zhen

    2017-10-01

    Multiple pharmacological applications of osthole have been previously recognized, including antioxidant, anti-inflammatory, anti‑platelet and estrogenic effects, and resistance to pain. The present study investigated the protective effects of osthole against inflammation in a rat model of chronic kidney failure (CRF) and the underlying mechanisms. Osthole treatment with significantly reversed CRF‑induced changes in serum creatinine, calcium, phosphorus and blood urea nitrogen levels in CRF rats. Male Sprague‑Dawley rats (age, 8 weeks) received 200 mg/kg 2% adenine suspension to induce CRF in the model group. In the osthole‑treated group, rats received 200 mg/kg 2% adenine suspension + osthole (40 mg/kg, intravenously). The results revealed that treatment with osthole significantly inhibited CRF‑induced tumor necrosis factor‑α, interleukin (IL)‑8 and IL‑6 expression, and suppressed nuclear factor‑κB (NF‑κB) protein expression in CRF rats. Osthole treatment significantly attenuated the protein expression of transforming growth factor‑β1 (TGF‑β1), reduced monocyte chemoattractant protein‑1 activity and increased the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) ratio in CRF rats. These results suggested that osthole protects against inflammation in a rat model of CRF via suppression of NF‑κB and TGF‑β1, and activation of PI3K/Akt/nuclear factor (erythroid‑derived 2)‑like 2 signaling. Therefore, osthole may represent a potential therapeutic agent for the treatment of CRF.

  2. Synthetic serine elastase inhibitor reduces cigarette smoke-induced emphysema in guinea pigs.

    PubMed

    Wright, Joanne L; Farmer, Stephen G; Churg, Andrew

    2002-10-01

    To test whether a serine elastase inhibitor could prevent or reduce emphysema, we exposed guinea pigs to cigarette smoke acutely, or daily for 6 months, and treated some animals with the neutrophil elastase inhibitor ZD0892. Acute smoke exposure increased lavage neutrophils and increased desmosine and hydroxyproline, measures of elastin and collagen breakdown; all these measures were reduced by ZD0892. Long-term smoke exposure produced emphysema and increases in lavage neutrophils, desmosine, hydroxyproline, and plasma tumor necrosis factor alpha (TNF-alpha). ZD0892 treatment returned lavage neutrophils, desmosine, and hydroxyproline levels to control values, and decreased airspace enlargement by 45% and TNF-alpha by 30%. Animals exposed to smoke for 4 months and then to smoke plus ZD0892 for 2 months were not protected against emphysema. Mice exposed to smoke showed increases in gene expression of neutrophil chemoattractant macrophage inflammatory protein-2, macrophage chemoattractant protein-1, and TNF-alpha at 2 hours along with increased plasma TNF-alpha; ZD0892 prevented the increases in macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 expression and reduced plasma TNF-alpha levels to baseline. These data demonstrate that a serine elastase inhibitor ameliorates the inflammatory and destructive effects of cigarette smoke, and that these effects are mediated in part by neutrophils and by smoke-driven TNF-alpha production.

  3. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    PubMed

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  4. Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare).

    PubMed

    Ligaba, Ayalew; Katsuhara, Maki; Shibasaka, Mineo; Djira, Gemechis

    2011-02-01

    In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  5. ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

    PubMed

    Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

    2007-11-01

    Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

  6. [Effect of qinghuobaiduyin on the expression of high mobility group box chromosomal protein 1 in macrophage].

    PubMed

    Li, Ping; Xu, Dan; Luo, Chengqun

    2010-07-01

    To observe the expression of high mobility group box chromosomal protein 1(HMGB1) in RAW264.7 macrophages after interfering with burning serum and qinghuobaidu-yin (QHBDY), and to find out the endogenous protection mechanism of QHBDY resisting inflammation reaction. RT-PCR was used to detect the expression of HMGB1 in RAW264.7 macrophages after interfering RAW264.7 macrophages with normal SD rat serum, burning SD rat serum, and QHBDY feeding SD rat serum. Small quantity of HMGB1 mRNA was expressed in RAW264.7. The expression of HMGB1 mRNA fluctuated around the standard level after interfering with normal serum of SD rats. The expression of HMGB1 mRNA rose at 3 h, and then decreased to the standard level; at 18 h, it rose rapidly; at 36 h, it reached the peak; and at 48 h, it remained at the high level after interfering with burning serum. The expression of HMGB1 mRNA increased at 3 h, and then decreased to the standard level. At 24 h, it started to rise after interfering with herb serum, and was lower than that of; the burning serum group (P<0.05). Burning serum can increase the expression of HMGB1 mRNA in RAW264.7. QHBDY can decrease the high expression of HMGB1 mRNA in RAW264.7 caused by burning serum.

  7. Improving membrane protein expression and function using genomic edits

    DOE PAGES

    Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

    2017-10-12

    Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

  8. Improving membrane protein expression and function using genomic edits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

    Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

  9. CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

    USGS Publications Warehouse

    Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

    2017-01-01

    Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to

  10. Statin-Induced Increases in Atrophy Gene Expression Occur Independently of Changes in PGC1α Protein and Mitochondrial Content

    PubMed Central

    Zacharewicz, Evelyn; Lee-Young, Robert S.; Snow, Rod J.; Russell, Aaron P.; McConell, Glenn K.

    2015-01-01

    One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg-1·day-1) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles. PMID:26020641

  11. Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes*

    PubMed Central

    Denhez, Benoit; Lizotte, Farah; Guimond, Marie-Odile; Jones, Nina; Takano, Tomoko; Geraldes, Pedro

    2015-01-01

    Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr1127 and Tyr1152 are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2+/C96Y) compared with control littermate mice (Ins2+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy. PMID:25404734

  12. Expression of the LIM-Homeodomain Protein Isl1 in the Developing and Mature Mouse Retina

    PubMed Central

    Elshatory, Yasser; Deng, Min; Xie, Xiaoling; Gan, Lin

    2010-01-01

    The mammalian retina is comprised of six major neuronal cell types and is subdivided into more morphological and physiological subtypes. The transcriptional machinery underlying these subtype fate choices is largely unknown. The LIM-homeodomain protein, Isl1, plays an essential role in central nervous system (CNS) differentiation but its relationship to retinal neurogenesis remains unknown. We report here its dynamic spatiotemporal expression in the mouse retina. Among bipolar interneurons, Isl1 expression commences at postnatal day (P)5 and is later restricted to ON-bipolar cells. The intensity of Isl1 expression is found to segregate the pool of ON-bipolar cells into rod and ON-cone bipolar cells with higher expression in rod bipolar cells. As bipolar cell development proceeds from P5–10 the colocalization of Isl1 and the pan-bipolar cell marker Chx10 reveals the organization of ON-center bipolar cell nuclei to the upper portion of the inner nuclear layer. Further, whereas Isl1 is predominantly a ganglion cell marker prior to embryonic day (E)15.5, at E15.5 and later its expression in nonganglion cells expands. We demonstrate that these Isl1-positive, nonganglion cells acquire the expression of amacrine cell markers embryonically, likely representing nascent cholinergic amacrine cells. Taken together, Isl1 is expressed during the maturation of and is later maintained in retinal ganglion cells and subtypes of amacrine and bipolar cells where it may function in the maintenance of these cells into adulthood. J. Comp. Neurol. 503: 182–197, 2007. PMID:17480014

  13. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    PubMed

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  14. Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

    PubMed Central

    Handisurya, Alessandra; Day, Patricia M.; Thompson, Cynthia D.; Buck, Christopher B.; Pang, Yuk-Ying S.; Lowy, Douglas R.

    2013-01-01

    Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism. PMID:24067981

  15. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    PubMed

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  16. Protein expression and genetic variability of canine Can f 1 in golden and Labrador retriever service dogs.

    PubMed

    Breitenbuecher, Christina; Belanger, Janelle M; Levy, Kerinne; Mundell, Paul; Fates, Valerie; Gershony, Liza; Famula, Thomas R; Oberbauer, Anita M

    2016-01-01

    Valued for trainability in diverse tasks, dogs are the primary service animal used to assist individuals with disabilities. Despite their utility, many people in need of service dogs are sensitive to the primary dog allergen, Can f 1, encoded by the Lipocalin 1 gene (LCN1). Several organizations specifically breed service dogs to meet special needs and would like to reduce allergenic potential if possible. In this study, we evaluated the expression of Can f 1 protein and the inherent variability of LCN1 in two breeds used extensively as service dogs. Saliva samples from equal numbers of male and female Labrador retrievers (n = 12), golden retrievers (n = 12), and Labrador-golden crosses (n = 12) were collected 1 h after the morning meal. Can f 1 protein concentrations in the saliva were measured by ELISA, and the LCN1 5' and 3' UTRs and exons sequenced. There was no sex effect (p > 0.2) nor time-of-day effect; however, Can f 1 protein levels varied by breed with Labrador retrievers being lower than golden retrievers (3.18 ± 0.51 and 5.35 ± 0.52 μg/ml, respectively, p < 0.0075), and the Labrador-golden crosses having intermediate levels (3.77 ± 0.48 μg/ml). Although several novel SNPs were identified in LCN1, there were no significant breed-specific sequence differences in the gene and no association of LCN1 genotypes with Can f 1 expression. As service dogs, Labrador retrievers likely have lower allergenic potential and, though there were no DNA sequence differences identified, classical genetic selection on the estimated breeding values associated with salivary Can f 1 expression may further reduce that potential.

  17. Keap1 knockdown increases markers of metabolic syndrome after long-term high fat diet feeding.

    PubMed

    More, Vijay R; Xu, Jialin; Shimpi, Prajakta C; Belgrave, Clyde; Luyendyk, James P; Yamamoto, Masayuki; Slitt, Angela L

    2013-08-01

    The nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway upregulates antioxidant and biotransformation enzyme expression to counter cellular oxidative stress. The contributions of Nrf2 to other cellular functions, such as lipid homeostasis, are emerging. This study was conducted to determine how enhanced Nrf2 activity influences the progression of metabolic syndrome with long-term high-fat diet (HFD) feeding. C57BL/6 and Keap1-knockdown (Keap1-KD) mice, which exhibit enhanced Nrf2 activity, were fed a HFD for 24 weeks. Keap1-KD mice had higher body weight and white adipose tissue mass compared to C57BL/6 mice on HFD, along with increased inflammation and lipogenic gene expression. HFD feeding increased hepatic steatosis and inflammation to a greater extent in Keap1-KD mice compared to C57BL/6 mice, which was associated with increased liver Cd36, fatty acid-binding protein 4, and monocyte chemoattractant protein 1 mRNA expression, as well as increased acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase-1 protein expression. The HFD altered short-term glucose homeostasis to a greater degree in Keap-KD mice compared to C57BL/6 mice, which was accompanied by downregulation of insulin receptor substrate 1 mRNA expression in skeletal muscle. Together, the results indicate that Keap1 knockdown, on treatment with HFD, increases certain markers of metabolic syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency.

    PubMed

    Jaber, Tareq; Henderson, Gail; Li, Sumin; Perng, Guey-Chuen; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

    2009-10-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5' end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5' terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection.

  19. Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals.

    PubMed

    Burnett, Lindsey A; Xiang, Xueyu; Bieber, Allan L; Chandler, Douglas E

    2008-01-01

    Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm. In X. tropicalis, allurin is coded for by the newly documented Crisp A gene. Recently, the observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals. Although an allurin gene has yet to be documented in mammals, Crisp proteins truncated post-translationally appear to exist in both the male and female reproductive tract of mammals.

  20. Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

    PubMed Central

    Roundhill, E; Burchill, S

    2013-01-01

    Background: Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. Methods: MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. Results: We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. Conclusion: For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy. PMID:23799853

  1. Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

    PubMed

    Roundhill, E; Burchill, S

    2013-07-09

    Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

  2. MyD88 expression in the rat dental follicle: Implications for osteoclastogenesis and tooth eruption

    PubMed Central

    Liu, Dawen; Yao, Shaomian; Wise, Gary E.

    2010-01-01

    Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin-1 (IL-1) and IL-18 Toll-like receptor signaling pathway. Because it is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine gene expression of MyD88 in vivo in the DFs from the first mandibular molars of postnatal rats from days 1–11. The results showed that MyD88 was expressed maximally at day 3. Using siRNA to knock down MyD88 expression in the DF cells also reduced the gene expression of nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1). IL-1α up-regulated the expression of NFKB1, MCP-1 and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1α effect. Conditioned medium from DF cells with MyD88 knocked down reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis as opposed to controls. In conclusion, the maximal expression of MyD88 at day 3 in the DF may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression. PMID:20662905

  3. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    PubMed Central

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O’Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5′untranslated region (UTR), a 285 base pair open reading frame (ORF) and a poly adenylation (A) signal (Holden et al., 1992 DNA Seq 3, 143-52). Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed. PMID:17306852

  4. Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

    PubMed

    Pathak, Bhakti R; Breed, Ananya A; Apte, Snehal; Acharya, Kshitish; Mahale, Smita D

    2016-01-01

    Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

  5. Pepsin-pancreatin protein hydrolysates from extruded amaranth inhibit markers of atherosclerosis in LPS-induced THP-1 macrophages-like human cells by reducing expression of proteins in LOX-1 signaling pathway

    PubMed Central

    2014-01-01

    Background Atherosclerosis is considered a progressive disease that affects arteries that bring blood to the heart, to the brain and to the lower end. It derives from endothelial dysfunction and inflammation, which play an important role in the thrombotic complications of atherosclerosis. Cardiovascular disease is the leading cause of death around the world and one factor that can contribute to its progression and prevention is diet. Our previous study found that amaranth hydrolysates inhibited LPS-induced inflammation in human and mouse macrophages by preventing activation of NF-κB signaling. Furthermore, extrusion improved the anti-inflammatory effect of amaranth protein hydrolysates in both cell lines, probably attributed to the production of bioactive peptides during processing. Therefore, the objective of this study was to compare the anti-atherosclerotic potential of pepsin-pancreatin hydrolysates from unprocessed and extruded amaranth in THP-1 lipopolysaccharide-induced human macrophages and suggest the mechanism of action. Results Unprocessed amaranth hydrolysate (UAH) and extruded amaranth hydrolysate (EAH) showed a significant reduction in the expression of interleukin-4 (IL-4) (69% and 100%, respectively), interleukin-6 (IL-6) (64% and 52%, respectively), interleukin-22 (IL-22) (55% and 70%, respectively). Likewise, UAH and EAH showed a reduction in the expression of monocyte-chemo attractant protein-1 (MCP-1) (35% and 42%, respectively), transferrin receptor-1 (TfR-1) (48% and 61%, respectively), granulocyte-macrophage colony-stimulating factor (GM-CSF) (59% and 63%, respectively), and tumor necrosis factor-α (TNF-α) (60% and 63%, respectively). Also, EAH reduced the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (27%), intracellular adhesion molecule-1 (ICAM-1) (28%) and matrix metalloproteinase-9 (MMP-9) (19%), important molecular markers in the atherosclerosis pathway. EAH, led to a reduction of 58, 52 and 79% for

  6. Expression of sigma receptor 1 mRNA and protein in rat retina.

    PubMed

    Liu, L L; Wang, L; Zhong, Y M; Yang, X L

    2010-06-02

    Sigma receptor (sigmaR), known as a unique nonopiate, nonphencyclidine brain receptor, can bind diverse classes of psychotropic drugs, neurosteroids and other synthetic compounds, such as (+)pentazocine, etc. Two types of sigmaRs have been identified: sigmaR1 and sigmaR2. In this work, we examined the expression of sigmaR1 in rat retina by reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling. RT-PCR analysis showed that sigmaR1 mRNA was present in rat retina. Furthermore, labeling for sigmaR1 was diffusely distributed in the outer and inner plexiform layers. The sigmaR1-immunoreactivity (IR) was also observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina sigmaR1 was expressed in all horizontal cells labeled by calbindin. In contrast, no sigmaR1-IR was detected in several subtypes of bipolar cells, including rod-dominant ON-type bipolar cells, types 2, 3, 5 and 8 bipolar cells, labeled by protein kinase C (PKC), recoverin and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) respectively. In the inner retina, most of GABAergic amacrine cells, including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, expressed sigmaR1. Some glycinergic amacrine cells were also labeled by sigmaR1, but glycinergic AII amacrine cells were not labeled. In addition, sigmaR1-IR was seen in almost all somata of the ganglion cells retrogradely labeled by fluorogold. These results suggest that sigmaR1 may have neuromodulatory and neuroprotective roles in the retina. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis

    PubMed Central

    Blanchard, Carine; Wang, Ning; Stringer, Keith F.; Mishra, Anil; Fulkerson, Patricia C.; Abonia, J. Pablo; Jameson, Sean C.; Kirby, Cassie; Konikoff, Michael R.; Collins, Margaret H.; Cohen, Mitchell B.; Akers, Rachel; Hogan, Simon P.; Assa’ad, Amal H.; Putnam, Philip E.; Aronow, Bruce J.; Rothenberg, Marc E.

    2006-01-01

    Eosinophilic esophagitis (EE) is an emerging disorder with a poorly understood pathogenesis. In order to define disease mechanisms, we took an empirical approach analyzing esophageal tissue by a genome-wide microarray expression analysis. EE patients had a striking transcript signature involving 1% of the human genome that was remarkably conserved across sex, age, and allergic status and was distinct from that associated with non-EE chronic esophagitis. Notably, the gene encoding the eosinophil-specific chemoattractant eotaxin-3 (also known as CCL26) was the most highly induced gene in EE patients compared with its expression level in healthy individuals. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia and mastocytosis. Furthermore, a single-nucleotide polymorphism in the human eotaxin-3 gene was associated with disease susceptibility. Finally, mice deficient in the eotaxin receptor (also known as CCR3) were protected from experimental EE. These results implicate eotaxin-3 as a critical effector molecule for EE and provide insight into disease pathogenesis. PMID:16453027

  8. Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

    PubMed

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

    2015-10-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

    PubMed Central

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

    2015-01-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

  10. FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

    PubMed

    Lungu, Gina; Covaleda, Lina; Mendes, Odete; Martini-Stoica, Heidi; Stoica, George

    2008-06-01

    Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK. (c) 2007 Wiley-Liss, Inc.

  11. Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP.

    PubMed

    Wong, Kah Keng; Ch'ng, Ewe Seng; Loo, Suet Kee; Husin, Azlan; Muruzabal, María Arestin; Møller, Michael B; Pedersen, Lars M; Pomposo, María Puente; Gaafar, Ayman; Banham, Alison H; Green, Tina M; Lawrie, Charles H

    2015-12-01

    Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (P<0.01 in all three datasets), and lower expression was correlated with worse overall survival (OS; P<0.01) and progression-free survival (PFS; P<0.05) in a microarray-profiled DLBCL dataset. At the protein level examined by immunohistochemistry, HIP1R expression at 30% cut-off was associated with GCB-DLBCL molecular subtype (P=0.0004; n=42), and predictive of OS (P=0.0006) and PFS (P=0.0230) in de novo DLBCL patients treated with R-CHOP (n=73). Cases with high FOXP1 and low HIP1R expression frequency (FOXP1(hi)/HIP1R(lo) phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1R<30% or FOXP1(hi)/HIP1R(lo) subgroup of patients exhibited inferior OS and PFS (P<0.05) independently of the International Prognostic Index. We conclude that HIP1R expression is strongly indicative of survival when utilized on its own or in combination with FOXP1, and the molecule is potentially applicable for subtyping of DLBCL cases. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

    PubMed

    Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

    2018-08-01

    To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. BAX protein expression and clinical outcome in epithelial ovarian cancer.

    PubMed

    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  14. The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

    PubMed

    Jang, Byeong-Churl

    2012-03-01

    High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

  15. Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

    PubMed

    Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

    2016-12-09

    Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Recombinant Human Sonic Hedgehog Protein Regulates the Expression of ZO-1 and Occludin by Activating Angiopoietin-1 in Stroke Damage

    PubMed Central

    Chen, Sheng-cai; Huang, Ming; Wang, Yong; Gao, Yuan; Huang, Yan; Wang, Meng-die; Mao, Ling; Hu, Bo

    2013-01-01

    This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage. PMID:23894369

  17. Emodin Increases Expression of Insulin-Like Growth Factor Binding Protein 1 through Activation of MEK/ERK/AMPKα and Interaction of PPARγ and Sp1 in Lung Cancer.

    PubMed

    Tang, Qing; Wu, JingJing; Zheng, Fang; Hann, Swei Sunny; Chen, YuQing

    2017-01-01

    Emodin has anti-neoplastic activities on multiple tumors. However, the molecular mechanisms underlying this effect still remain to be fully understood. Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays and flow cytometry, respectively. Cell invasion and migration were examined by transwell invasion and wound healing assays. Western blot analysis was performed to examine the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα), extracellular signaling-regulated kinase 1/2 (ERK1/2), peroxisome proliferators-activated receptor gamma (PPARγ), insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and the transcription factor Sp1. QRT-PCR was used to examine the mRNA levels of the IGFBP1 gene. Small interfering RNAs (siRNAs) were used to knockdown PPARγ and IGFBP1 genes. Exogenously expression of IGFBP1 and Sp1 was determined by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings. We showed that emodin induced cell cycle arrest of NSCLC cells. Emodin increased PPARγ protein and luciferase reporter activity, which were abolished by inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK and AMPK. Silencing of PPARγ abrogated emodin-inhibited cell growth and cell cycle arrest. Furthermore, emodin elevated IGFBP1 mRNA, protein, and promoter activity through activation of PPARγ. Intriguingly, overexpressed Sp1 attenuated emodin-induced IGFBP1 expression, which was not observed in cells with silenced PPARγ gene. Moreover, silencing of IGFBP1 gene blunted emodin-induced inhibition of cell growth and cell cycle arrest. On the contrary, overexpressed IGFBP1 enhanced emodin-induced phosphorylation of AMPKα and ERK1/2, and restored emodin-inhibited growth in

  18. Expression of programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) in the tumor microenvironment and in tumor-draining lymph nodes of breast cancer.

    PubMed

    Ye, Qian; Wang, Chenglong; Xian, Jie; Zhang, Ming; Cao, Yijia; Cao, Youde

    2018-05-01

    Programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) are both immunosuppressive proteins. Here, we investigated the relationship between PD-1 and IDO in the tumor microenvironment (TME) and in tumor-draining lymph nodes (TDLNs) in breast cancer patients. First, the protein and mRNA expression levels of PD-1 and IDO in 20 frozen tissues were examined using Western blotting and real-time polymerase chain reaction. Second, 151 paraffin-embedded breast samples and 52 lymph node samples were analyzed by immunohistochemistry. Third, correlation and survival data for PD-1 and IDO in 963 breast tumor patients were mined using the cBio Cancer Genomics Portal. We found that the protein expression level of IDO was significantly increased in frozen tumor tissues (P = .005). From paraffin-embedded samples in the TME, PD-1 + cells were only located in the stroma, while IDO was expressed in myoepithelial, stromal, and tumor cells. PD-1 and stromal IDO in the TME showed increased expression in tumors (P< .001 and P < .001, respectively). In TDLNs, PD-1 + cells were primarily located in the germinal centers (GCs), and IDO + cells were primarily located in the paracortex. Normal lymph nodes expressed PD-1 and IDO at the same level as non-metastatic and metastatic lymph nodes (P = .151 and P = .812, respectively). According to cBioPortal, the correlation analysis showed that IDO and PD-1 had high correlation coefficients (r = 0.83). These findings suggest that there is a positive correlation between the expression of PD-1 and IDO and that blocking both PD-1 and IDO pathways may represent an attractive therapeutic strategy in breast cancer treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Increased expression of high mobility group box protein 1 and vascular endothelial growth factor in placenta previa.

    PubMed

    Xie, Han; Qiao, Ping; Lu, Yi; Li, Ying; Tang, Yuping; Huang, Yiying; Bao, Yirong; Ying, Hao

    2017-12-01

    Placenta previa is often associated with preterm delivery, reduced birth weight, a higher frequency of placental accreta and postpartum haemorrhage, and increased likelihood of blood transfusion. The present study aimed to examine the expression of high mobility group box protein 1 (HMGB1) in the placenta of women with or without placenta previa. The study group consisted of placental tissues obtained from women with or without placenta previa. The expression levels of HMGB1 and vascular endothelial growth factor (VEGF) were evaluated in the placental tissues using reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry. The mRNA expression levels of HMGB1 and VEGF were significantly increased in the placenta previa group compared with in the normal group. In addition, the placenta previa group exhibited increased HMGB1 and VEGF staining in vascular endothelial cells and trophoblasts. There were no significant differences in the expression of HMGB1 or VEGF between groups with or without placenta accreta or postpartum haemorrhage. The present study hypothesised that the increased expression of HMGB1 in the placenta may be associated with the pathogenesis of placenta previa by regulating the expression of the proangiogenic factor VEGF.

  20. Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.

    PubMed

    Kalapothakis, Evanguedes; Araujo, Simone Costa; de Castro, Cibele Soares; Mendes, Thais Melo; Gomez, Marcus Vinícius; Mangili, Oldemir C; Gubert, Ida C; Chávez-Olórtegui, Carlos

    2002-12-01

    The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3' region with 218 nucleotides. LiD1 was expressed as a protein fused with beta-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.

  1. Enhanced expression of Gqα and PLC-β1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1.

    PubMed

    Atef, Mohammed Emehdi; Anand-Srivastava, Madhu B

    2014-07-01

    Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy. Copyright © 2014 the American Physiological Society.

  2. Antibody responses to prime-boost vaccination with an HIV-1 gp145 envelope protein and chimpanzee adenovirus vectors expressing HIV-1 gp140.

    PubMed

    Emmer, Kristel L; Wieczorek, Lindsay; Tuyishime, Steven; Molnar, Sebastian; Polonis, Victoria R; Ertl, Hildegund C J

    2016-10-23

    Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate. Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups. Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay. Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein. Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies.

  3. Protective Effect of Tropisetron on Rodent Hepatic Injury after Trauma-Hemorrhagic Shock through P38 MAPK-Dependent Hemeoxygenase-1 Expression

    PubMed Central

    Hwang, Tsong-Long; Tsai, Yung-Fong

    2012-01-01

    Tropisetron can decrease inflammatory cell responses and alleviate organ damage caused by trauma-hemorrhage, but the mechanism of these effects remains unknown. The p38 mitogen-activated protein kinase/hemeoxygenase-1 (p38 MAPK/HO-1) pathway exerts anti-inflammatory effects on different tissues. The aim of this study was to investigate whether p38 MAPK/HO-1 plays any role in the tropisetron-mediated attenuation of hepatic injury after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure maintained at approximately 35–40 mmHg for 90 min), followed by fluid resuscitation. During resuscitation, several treatment regimens were administered: four doses of tropisetron alone (0.1, 0.3, 1, 3 mg/kg body weight), or a single dose of tropisetron (1 mg/kg body weight) with and without a p38 MAPK inhibitor (SB-203580, 2 mg/kg body weight) or HO antagonist (chromium-mesoporphyrin, 2.5 mg/kg body weight). Various parameters were measured, and the animals were sacrificed at 24 h post-resuscitation. The results showed that trauma-hemorrhage increased the following parameters: plasma concentrations of aspartate (AST) and alanine aminotransferases (ALT), hepatic myeloperoxidase (MPO) activity, and levels of cytokine-induced neutrophil chemoattractant-1 and -3 (CINC-1 and CINC-3), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-1α (MIP-1α). These parameters were significantly improved in the tropisetron-treated rats subjected to trauma-hemorrhage. Tropisetron treatment also increased hepatic p38 MAPK and HO-1 expression compared with vehicle-treated trauma-hemorrhaged rats. Co-administration of SB-203580 or chromium-mesoporphyrin with tropisetron abolished the tropisetron-induced beneficial effects on the above parameters and hepatic injury. These results suggest that the protective effect of tropisetron administration on alleviation of hepatic injury

  4. Protective effect of tropisetron on rodent hepatic injury after trauma-hemorrhagic shock through P38 MAPK-dependent hemeoxygenase-1 expression.

    PubMed

    Liu, Fu-Chao; Yu, Huang-Ping; Hwang, Tsong-Long; Tsai, Yung-Fong

    2012-01-01

    Tropisetron can decrease inflammatory cell responses and alleviate organ damage caused by trauma-hemorrhage, but the mechanism of these effects remains unknown. The p38 mitogen-activated protein kinase/hemeoxygenase-1 (p38 MAPK/HO-1) pathway exerts anti-inflammatory effects on different tissues. The aim of this study was to investigate whether p38 MAPK/HO-1 plays any role in the tropisetron-mediated attenuation of hepatic injury after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure maintained at approximately 35-40 mmHg for 90 min), followed by fluid resuscitation. During resuscitation, several treatment regimens were administered: four doses of tropisetron alone (0.1, 0.3, 1, 3 mg/kg body weight), or a single dose of tropisetron (1 mg/kg body weight) with and without a p38 MAPK inhibitor (SB-203580, 2 mg/kg body weight) or HO antagonist (chromium-mesoporphyrin, 2.5 mg/kg body weight). Various parameters were measured, and the animals were sacrificed at 24 h post-resuscitation. The results showed that trauma-hemorrhage increased the following parameters: plasma concentrations of aspartate (AST) and alanine aminotransferases (ALT), hepatic myeloperoxidase (MPO) activity, and levels of cytokine-induced neutrophil chemoattractant-1 and -3 (CINC-1 and CINC-3), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-1α (MIP-1α). These parameters were significantly improved in the tropisetron-treated rats subjected to trauma-hemorrhage. Tropisetron treatment also increased hepatic p38 MAPK and HO-1 expression compared with vehicle-treated trauma-hemorrhaged rats. Co-administration of SB-203580 or chromium-mesoporphyrin with tropisetron abolished the tropisetron-induced beneficial effects on the above parameters and hepatic injury. These results suggest that the protective effect of tropisetron administration on alleviation of hepatic injury

  5. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    PubMed Central

    2009-01-01

    Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant

  6. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

    PubMed

    Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria Elena; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello; Marusic, Carla

    2009-11-20

    In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with

  7. Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

    PubMed

    Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

    2013-01-01

    Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation

  8. Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

    PubMed Central

    Lechner, F; Machado, J; Bertoni, G; Seow, H F; Dobbelaere, D A; Peterhans, E

    1997-01-01

    Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of

  9. Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells.

    PubMed

    Ozgun, Eray; Sayilan Ozgun, Gulben; Tabakcioglu, Kiymet; Suer Gokmen, Selma; Sut, Necdet; Eskiocak, Sevgi

    2017-10-01

    Paraoxonase-1 (PON1) and PON3 (PON3) are anti-atherosclerotic enzymes, synthesized primarily in liver and bound to HDL in circulation. The aim of the present study was to investigate the effects of therapeutic doses of lipoic acid on PON1 and PON3 protein levels, mRNA expression and arylesterase activity in liver. We treated HepG2 cells with 10, 40 and 200 μM lipoic acid for 72 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PON1 and PON3 protein levels were measured by Western blotting, their mRNA expression was measured by quantitative PCR and arylesterase activity was measured spectrophotometrically. 200 µM lipoic acid caused a significant increase on PON1 and PON3 protein levels and arylesterase activity as compared with control, 10 µM and 40 µM lipoic acid-treated cells. 200 µM lipoic acid also caused a significant decrease on PON1 mRNA expression whereas on a significant increase PON3 mRNA expression as compared with control, 10 µM and 40 µM lipoic acid-treated cells. Our study showed that although lipoic acid up-regulates PON3 but down-regulates PON1 mRNA expression, it increases both PON1 and PON3 protein levels and arylesterase activity in HepG2 cells. We can report that lipoic acid may be useful for preventing atherosclerosis at therapeutic doses.

  10. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    PubMed Central

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  11. [Tripartite-motif protein 25 and pyruvate kinase M2 protein expression in non-small cell lung cancer].

    PubMed

    Jing, Huai-Zhi; Qiu, Feng; Chen, Shi-Zhi; Su, Lin; Qu, Can

    2015-03-01

    To investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC. The expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients. The positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026). TRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

  12. The E4 protein; structure, function and patterns of expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genomemore » amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1

  13. Protein Kinase WNK1 Promotes Cell Surface Expression of Glucose Transporter GLUT1 by Regulating a Tre-2/USP6-BUB2-Cdc16 Domain Family Member 4 (TBC1D4)-Rab8A Complex*

    PubMed Central

    Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Jordan, Peter

    2010-01-01

    One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. PMID:20937822

  14. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  15. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

    PubMed

    Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

    2015-01-21

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

  16. TPPII, MYBBP1A and CDK2 form a protein-protein interaction network.

    PubMed

    Nahálková, Jarmila; Tomkinson, Birgitta

    2014-12-15

    Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

    PubMed

    Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

    2016-10-01

    Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

  18. EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells.

    PubMed

    Jiang, Jun-xia; Zhang, Shui-juan; Xiong, Yao-kang; Jia, Yong-liang; Sun, Yan-hong; Lin, Xi-xi; Shen, Hui-juan; Xie, Qiang-min; Yan, Xiao-feng

    2015-01-01

    Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.

  19. EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

    PubMed Central

    Xiong, Yao-kang; Jia, Yong-liang; Sun, Yan-hong; Lin, Xi-xi; Shen, Hui-juan; Xie, Qiang-min; Yan, Xiao-feng

    2015-01-01

    Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells. PMID:26035589

  20. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

    PubMed

    Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

    2015-05-01

    Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Protein-protein interaction network of gene expression in the hydrocortisone-treated keloid.

    PubMed

    Chen, Rui; Zhang, Zhiliang; Xue, Zhujia; Wang, Lin; Fu, Mingang; Lu, Yi; Bai, Ling; Zhang, Ping; Fan, Zhihong

    2015-01-01

    In order to explore the molecular mechanism of hydrocortisone in keloid tissue, the gene expression profiles of keloid samples treated with hydrocortisone were subjected to bioinformatics analysis. Firstly, the gene expression profiles (GSE7890) of five samples of keloid treated with hydrocortisone and five untreated keloid samples were downloaded from the Gene Expression Omnibus (GEO) database. Secondly, data were preprocessed using packages in R language and differentially expressed genes (DEGs) were screened using a significance analysis of microarrays (SAM) protocol. Thirdly, the DEGs were subjected to gene ontology (GO) function and KEGG pathway enrichment analysis. Finally, the interactions of DEGs in samples of keloid treated with hydrocortisone were explored in a human protein-protein interaction (PPI) network, and sub-modules of the DEGs interaction network were analyzed using Cytoscape software. Based on the analysis, 572 DEGs in the hydrocortisone-treated samples were screened; most of these were involved in the signal transduction and cell cycle. Furthermore, three critical genes in the module, including COL1A1, NID1, and PRELP, were screened in the PPI network analysis. These findings enhance understanding of the pathogenesis of the keloid and provide references for keloid therapy. © 2015 The International Society of Dermatology.

  2. Effects of fibroblast growth factor-2 on the expression and regulation of chemokines in human dental pulp cells.

    PubMed

    Kim, Young-Suk; Min, Kyung-San; Jeong, Dong-Ho; Jang, Jun-Hyeog; Kim, Hae-Won; Kim, Eun-Cheol

    2010-11-01

    Fibroblast growth factor-2 (FGF-2) participates in both hematopoiesis and osteogenesis; however, the effects of FGF-2 on chemokines during odontoblastic differentiation have not been reported. This study investigated whether human dental pulp cells (HDPCs) treated with FGF-2 could express chemokines during differentiation into odontoblastic cells and sought to identify its underlying mechanism of action. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, calcified nodule formation by alizarin red staining, and marker RNA (mRNA) expression by reverse-transcriptase polymerase chain reaction (RT-PCR). Expression of chemokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and MIP-3α, were evaluated by RT-PCR. ALP activity, the mineralization, and mRNA expression for odontoblastic markers were enhanced by FGF-2 in HDPCs. FGF-2 also up-regulated the expression of IL-6, IL-8, MCP-1, MIP-1α, and MIP-3α mRNAs, which were attenuated by inhibitors of p38, ERK1/2 and p38 MAP kinases, protein kinase C, phosphoinositide-3 kinase, and NF-κB. Taken together, these data suggest that FGF-2 plays a role not only as a differentiation inducing factor in the injury repair processes of pulpal tissue but also as a positive regulator of chemokine expression, which may help in tissue engineering and pulp regeneration using HDPCs. However, the fate of odontoblastic or osteoblastic differentiation, effective local delivery for FGF-2, interaction of chemotatic and odontogenic factors, and other limitations will need to be overcome before a major modality for the treatment of pulp disease. Copyright © 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. Differential expression of steroidogenic factors 1 and 2, cytochrome p450scc, and steroidogenic acute regulatory protein in human pancreas.

    PubMed

    Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Morimoto, Sumiko; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

    2008-08-01

    The aim of this study was to investigate the expression of the 4 gene transcripts, steroidogenic factors 1 (SF-1) and 2 (SF-2), steroidogenic acute regulatory (StAR), and cytochrome P450 11A1, involved in the synthesis of steroid hormones in normal human pancreas. Total RNA was extracted from normal male (n = 5) and female (n = 5) samples, obtained from the organ donor program. The expression levels of SF-1, SF-2, StAR protein, and P450scc were assessed by reverse transcription-polymerase chain reaction and complemented with immunohistochemistry analysis. Polymerase chain reaction products amplification for all genes was present in both male and female samples, although differential expression was observed. The signals detected were much more evident in male than in female messenger RNA isolates for SF-1, SF-2, and StAR protein. The expression for P450scc was more intense in female samples. A similar pattern was observed in the immunohistochemical studies. Normal human pancreas expresses 4 gene transcripts involved in steroid synthesis similarly to steroidogenic organs. A distinctive characteristic is the sexually dimorphic expression of these factors. These data provide further evidence to support that the pancreas is a truly steroidogenic tissue, highlighting the presence of sex- and location-related differences in the expression of steroidogenic factors.

  4. Acute exercise increases brain region-specific expression of MCT1, MCT2, MCT4, GLUT1, and COX IV proteins.

    PubMed

    Takimoto, Masaki; Hamada, Taku

    2014-05-01

    The brain is capable of oxidizing lactate and ketone bodies through monocarboxylate transporters (MCTs). We examined the protein expression of MCT1, MCT2, MCT4, glucose transporter 1 (GLUT1), and cytochrome-c oxidase subunit IV (COX IV) in the rat brain within 24 h after a single exercise session. Brain samples were obtained from sedentary controls and treadmill-exercised rats (20 m/min, 8% grade). Acute exercise resulted in an increase in lactate in the cortex, hippocampus, and hypothalamus, but not the brainstem, and an increase in β-hydroxybutyrate in the cortex alone. After a 2-h exercise session MCT1 increased in the cortex and hippocampus 5 h postexercise, and the effect lasted in the cortex for 24 h postexercise. MCT2 increased in the cortex and hypothalamus 5-24 h postexercise, whereas MCT2 increased in the hippocampus immediately after exercise, and remained elevated for 10 h postexercise. Regional upregulation of MCT2 after exercise was associated with increases in brain-derived neurotrophic factor and tyrosine-related kinase B proteins, but not insulin-like growth factor 1. MCT4 increased 5-10 h postexercise only in the hypothalamus, and was associated with increased hypoxia-inducible factor-1α expression. However, none of the MCT isoforms in the brainstem was affected by exercise. Whereas GLUT 1 in the cortex increased only at 18 h postexercise, COX IV in the hippocampus increased 10 h after exercise and remained elevated for 24 h postexercise. These results suggest that acute prolonged exercise induces the brain region-specific upregulation of MCT1, MCT2, MCT4, GLUT1, and COX IV proteins.

  5. The Expression and Significance of Neuronal Iconic Proteins in Podocytes

    PubMed Central

    Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

    2014-01-01

    Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

  6. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    PubMed Central

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  7. Improving membrane protein expression by optimizing integration efficiency

    PubMed Central

    2017-01-01

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

  8. The gene expression of cytokines and chemokines induced by tourniquet shock in mice.

    PubMed

    Tanaka, Jin; Ishida, Yuko; Ohshima, Tohru; Kondo, Toshikazu

    2003-09-01

    Traumatic shock is one of the major fields in forensic pathology, but its mechanism remains elusive from the pathophysiological aspects. Tourniquet shock has been established as one of the animal models of traumatic shock, and we examined the gene expression of cytokines and chemokines in the lung and liver in tourniquet shock using mice. Tourniquet was conducted by the application of elastic bands with five turns at both the thighs as high as possible for 2 h, followed by reperfusion. In this procedure, more than 90% mice died within 48 h after reperfusion. Serum hepatic transaminase and hematocrit values significantly increased at 2 h after reperfusion, and their elevation was still evident after 10 h. Histopathologically, hemorrhages, congestion and leukocyte recruitment were observed in the lung and liver specimens after 6 h of reperfusion. Immunohistochemical analysis with anti-myeloperoxidase antibody demonstrated a massive neutrophil infiltration in the lung and liver at 2 h or more after reperfusion. RT-PCR analyses demonstrated that the gene expression of interleukin-1beta, tumor necrosis factor-alpha, monocytes chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-2, KC and vascular endothelial adhesion molecule-1 was most enhanced in the lung and liver at 2 h after reperfusion. Thus, the gene expression of cytokines and chemokines is presumed to be closely related with the onset of tourniquet shock. From the forensic aspects, these cytokines and chemokines are considered to be useful markers for the early diagnosis of tourniquet shock.

  9. Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

    PubMed

    Chee Wei, T; Nurul Wahida, A G; Shaharum, S

    2014-12-01

    Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

  10. Circulating monocyte chemoattractant protein‐1 (MCP‐1) is associated with cachexia in treatment‐naïve pancreatic cancer patients

    PubMed Central

    Talbert, Erin E.; Lewis, Heather L.; Farren, Matthew R.; Ramsey, Mitchell L.; Chakedis, Jeffery M.; Rajasekera, Priyani; Haverick, Ericka; Sarna, Angela; Bloomston, Mark; Pawlik, Timothy M.; Zimmers, Teresa A.; Lesinski, Gregory B.; Hart, Phil A.; Dillhoff, Mary E.; Schmidt, Carl R.

    2018-01-01

    Abstract Background Cancer‐associated wasting, termed cancer cachexia, has a profound effect on the morbidity and mortality of cancer patients but remains difficult to recognize and diagnose. While increases in circulating levels of a number of inflammatory cytokines have been associated with cancer cachexia, these associations were generally made in patients with advanced disease and thus may be associated with disease progression rather than directly with the cachexia syndrome. Thus, we sought to assess potential biomarkers of cancer‐induced cachexia in patients with earlier stages of disease. Methods A custom multiplex array was used to measure circulating levels of 25 soluble factors from 70 pancreatic cancer patients undergoing attempted tumour resections. A high‐sensitivity multiplex was used for increased sensitivity for nine cytokines. Results Resectable pancreatic cancer patients with cachexia had low levels of canonical pro‐inflammatory cytokines including interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β), interferon‐γ (IFN‐γ), and tumour necrosis factor (TNF). Even in our more sensitive analysis, these cytokines were not associated with cancer cachexia. Of the 25 circulating factors tested, only monocyte chemoattractant protein‐1 (MCP‐1) was increased in treatment‐naïve cachectic patients compared with weight stable patients and identified as a potential biomarker for cancer cachexia. Although circulating levels of leptin and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were found to be decreased in the same cohort of treatment‐naïve cachectic patients, these factors were closely associated with body mass index, limiting their utility as cancer cachexia biomarkers. Conclusions Unlike in advanced disease, it is possible that cachexia in patients with resectable pancreatic cancer is not associated with high levels of classical markers of systemic inflammation. However, cachectic, treatment‐naïve patients

  11. Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters*

    PubMed Central

    Giral, Hector; Lanzano, Luca; Caldas, Yupanqui; Blaine, Judith; Verlander, Jill W.; Lei, Tim; Gratton, Enrico; Levi, Moshe

    2011-01-01

    The sodium-dependent phosphate (Na/Pi) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of Pi. The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in Pi reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/Pi transporters. Pdzk1−/− mice adapted to chronic low Pi diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/Pi transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/Pi transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low Pi concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/Pi transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c. PMID:21388960

  12. Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

    PubMed

    Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

    2018-02-01

    Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

  13. An update on the LIM and SH3 domain protein 1 (LASP1): a versatile structural, signaling, and biomarker protein

    PubMed Central

    Orth, Martin F.; Cazes, Alex; Butt, Elke; Grunewald, Thomas G. P.

    2015-01-01

    The gene encoding the LIM and SH3 domain protein (LASP1) was cloned two decades ago from a cDNA library of breast cancer metastases. As the first protein of a class comprising one N-terminal LIM and one C-terminal SH3 domain, LASP1 founded a new LIM-protein subfamily of the nebulin group. Since its discovery LASP1 proved to be an extremely versatile protein because of its exceptional structure allowing interaction with various binding partners, its ubiquitous expression in normal tissues, albeit with distinct expression patterns, and its ability to transmit signals from the cytoplasm into the nucleus. As a result, LASP1 plays key roles in cell structure, physiological processes, and cell signaling. Furthermore, LASP1 overexpression contributes to cancer aggressiveness hinting to a potential value of LASP1 as a cancer biomarker. In this review we summarize published data on structure, regulation, function, and expression pattern of LASP1, with a focus on its role in human cancer and as a biomarker protein. In addition, we provide a comprehensive transcriptome analysis of published microarrays (n=2,780) that illustrates the expression profile of LASP1 in normal tissues and its overexpression in a broad range of human cancer entities. PMID:25622104

  14. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  15. Expression of gamma-aminobutyric acid rho 1 and rho 1 Delta 450 as gene fusions with the green fluorescent protein.

    PubMed

    Martinez-Torres, A; Miledi, R

    2001-02-13

    The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type rho 1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, rho 1 Delta 450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

  16. Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein

    PubMed Central

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2001-01-01

    The functional characteristics and cellular localization of the γaminobutyric acid (GABA) ρ1 receptor and its nonfunctional isoform ρ1Δ450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type ρ1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, ρ1Δ450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that ρ1Δ450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors. PMID:11172056

  17. Expression of Water Channel Proteins in Mesembryanthemum crystallinum1

    PubMed Central

    Kirch, Hans-Hubert; Vera-Estrella, Rosario; Golldack, Dortje; Quigley, Francoise; Michalowski, Christine B.; Barkla, Bronwyn J.; Bohnert, Hans J.

    2000-01-01

    We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux. PMID:10806230

  18. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    PubMed

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

  19. Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat. Potential role of adiponectin receptor 1

    PubMed Central

    Sideri, Aristea; Stavrakis, Dimitris; Bowe, Collin; Shih, David Q.; Fleshner, Phillip; Arsenescu, Violeta; Arsenescu, Razvan; Turner, Jerrold R.; Pothoulakis, Charalabos

    2015-01-01

    In inflammatory bowel disease (IBD), obesity is associated with worsening of the course of disease. Here, we examined the role of obesity in the development of colitis and studied mesenteric fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity with the trinitrobenzene sulfonic acid (TNBS) colitis mouse model to create groups with obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed by histology, myeloperoxidase assay, and cytokine mRNA expression by real-time PCR. Medium from human mesenteric fat and cultured preadipocytes was obtained from obese patients and those with IBD. Histological analysis showed inflammatory cell infiltrate and increased histological damage in the intestine and mesenteric fat of obese mice with colitis compared with all other groups. Obesity also increased the expression of proinflammatory cytokines including IL-1β, TNF-α, monocyte chemoattractant protein 1, and keratinocyte-derived chemokine, while it decreased the TNBS-induced increases in IL-2 and IFN-γ in mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese patients and those with and IBD demonstrated differential release of adipokines and growth factors compared with controls. Fat-conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460 colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced colitis. In conclusion, obesity worsens the outcome of experimental colitis, and obesity- and IBD-associated changes in adipose tissue promote differential mediator release in mesenteric fat that modulates colonocyte responses and may affect the course of colitis. Our results also suggest an important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during colitis. PMID:25591865

  20. Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

    PubMed

    Borecký, J; Maia, I G; Costa, A D; Jezek, P; Chaimovich, H; de Andrade, P B; Vercesi, A E; Arruda, P

    2001-09-14

    The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

  1. Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

    PubMed

    Chen, Shulian; Peng, Chuandu; Wei, Xin; Luo, Deyi; Lin, Yifei; Yang, Tongxin; Jin, Xi; Gong, Lina; Li, Hong; Wang, Kunjie

    2017-08-01

    To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

  2. Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

    PubMed

    Ansardi, D C; Porter, D C; Morrow, C D

    1991-04-01

    The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

  3. Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

    PubMed Central

    2012-01-01

    Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981

  4. Structural insights into the interaction between a potent anti-inflammatory protein, viral CC chemokine inhibitor (vCCI), and the human CC chemokine, Eotaxin-1.

    PubMed

    Kuo, Nai-Wei; Gao, Yong-Guang; Schill, Megan S; Isern, Nancy; Dupureur, Cynthia M; Liwang, Patricia J

    2014-03-07

    Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

  5. Structural Insights into the Interaction between a Potent Anti-inflammatory Protein, Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine, Eotaxin-1*

    PubMed Central

    Kuo, Nai-Wei; Gao, Yong-Guang; Schill, Megan S.; Isern, Nancy; Dupureur, Cynthia M.; LiWang, Patricia J.

    2014-01-01

    Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines. PMID:24482230

  6. Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

    PubMed

    Hsieh, C M; Fukumoto, S; Layne, M D; Maemura, K; Charles, H; Patel, A; Perrella, M A; Lee, M E

    2000-11-24

    Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

  7. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  8. Placental Expressions of CDKN1C and KCNQ1OT1 in Monozygotic Twins with Selective Intrauterine Growth Restriction.

    PubMed

    Gou, Chenyu; Liu, Xiangzhen; Shi, Xiaomei; Chai, Hanjing; He, Zhi-Ming; Huang, Xuan; Fang, Qun

    2017-10-01

    CDKN1C and KCNQ1OT1 are imprinted genes that might be potential regulators of placental development. This study investigated placental expressions of CDKN1C and KCNQ1OT1 in monozygotic twins with and without selective intrauterine growth restriction (sIUGR). Seventeen sIUGR and fifteen normal monozygotic(MZ) twin pairs were examined. Placental mRNA expressions of CDKN1C and KCNQ1OT1 were detected by real-time fluorescent quantitative PCR. CDKN1C protein expression was detected by immunohistochemical assay and Western-blotting. In the sIUGR group, smaller fetuses had a smaller share of the placenta, and CDKN1C protein expression was significantly increased while KCNQ1OT1 mRNA expression was significantly decreased. The CDKN1C/KCNQ1OT1 mRNA ratio was lower in the larger fetus than in the smaller fetus (p < .05). In the control group, CDKN1C protein expression showed no difference between larger and smaller fetuses, while KCNQ1OT1 mRNA expression was significantly lower in the larger fetus, and the CDKN1C/KCNQ1OT1 mRNA ratio was higher in the larger fetus than in the smaller fetus (p < .05). Our findings showed that pathogenesis of sIUGR may be related to the co-effect of the up-regulated protein expression of CDKN1C and down-regulated mRNA expression of KCNQ1OT1 in the placenta.

  9. CD274/PD-L1 gene amplification and PD-L1 protein expression are common events in squamous cell carcinoma of the oral cavity.

    PubMed

    Straub, Melanie; Drecoll, Enken; Pfarr, Nicole; Weichert, Wilko; Langer, Rupert; Hapfelmeier, Alexander; Götz, Carolin; Wolff, Klaus-Dietrich; Kolk, Andreas; Specht, Katja

    2016-03-15

    Immunomodulatory therapies, targeting the immune checkpoint receptor-ligand complex PD-1/PD-L1 have shown promising results in early phase clinical trials in solid malignancies, including carcinomas of the head and neck. In this context, PD-L1 protein expression has been proposed as a potentially valuable predictive marker. In the present study, expression of PD-L1 and PD-1 was evaluated by immunohistochemistry in 80 patients with predominantly HPV-negative oral squamous cell carcinomas and associated nodal metastasis. In addition, CD274/PD-L1 gene copy number status was assessed by fluorescence in situ hybridization analysis. PD-L1 expression was detected in 36/80 (45%) cases and concordance of PD-L1 expression in primary tumor and corresponding nodal metastasis was present in only 20/28 (72%) cases. PD-1 expression was found in tumor-infiltrating lymphocytes (TILs) but not in tumor cells. CD274/PD-L1 gene amplification was detected in 19% of cases, with high level PD-L1 amplification present in 12/80 (15%), and low level amplification in 3/80 (4%). Interestingly, CD274/PD-L1 gene amplification was associated with positive PD-L1 immunostaining in only 73% of cases. PD-L1 copy number status was concordant in primary tumor and associated metastases. Clinically, PD-L1 tumor immunopositivity was associated with a higher risk for nodal metastasis at diagnosis, overall tumor related death und recurrence. Based on our findings we propose to include PD-L1 copy number status in addition to protein status in screening programs for future clinical trials with immunotherapeutic strategies targeting the PD-1/PD-L1 axis.

  10. CD274/PD-L1 gene amplification and PD-L1 protein expression are common events in squamous cell carcinoma of the oral cavity

    PubMed Central

    Straub, Melanie; Drecoll, Enken; Pfarr, Nicole; Weichert, Wilko; Langer, Rupert; Hapfelmeier, Alexander; Götz, Carolin; Wolff, Klaus-Dietrich; Kolk, Andreas; Specht, Katja

    2016-01-01

    Immunomodulatory therapies, targeting the immune checkpoint receptor-ligand complex PD-1/PD-L1 have shown promising results in early phase clinical trials in solid malignancies, including carcinomas of the head and neck. In this context, PD-L1 protein expression has been proposed as a potentially valuable predictive marker. In the present study, expression of PD-L1 and PD-1 was evaluated by immunohistochemistry in 80 patients with predominantly HPV-negative oral squamous cell carcinomas and associated nodal metastasis. In addition, CD274/PD-L1 gene copy number status was assessed by fluorescence in situ hybridization analysis. PD-L1 expression was detected in 36/80 (45%) cases and concordance of PD-L1 expression in primary tumor and corresponding nodal metastasis was present in only 20/28 (72%) cases. PD-1 expression was found in tumor-infiltrating lymphocytes (TILs) but not in tumor cells. CD274/PD-L1 gene amplification was detected in 19% of cases, with high level PD-L1 amplification present in 12/80 (15%), and low level amplification in 3/80 (4%). Interestingly, CD274/PD-L1 gene amplification was associated with positive PD-L1 immunostaining in only 73% of cases. PD-L1 copy number status was concordant in primary tumor and associated metastases. Clinically, PD-L1 tumor immunopositivity was associated with a higher risk for nodal metastasis at diagnosis, overall tumor related death und recurrence. Based on our findings we propose to include PD-L1 copy number status in addition to protein status in screening programs for future clinical trials with immunotherapeutic strategies targeting the PD-1/PD-L1 axis. PMID:26918453

  11. Prostaglandin H2 induces the migration of human eosinophils through the chemoattractant receptor homologous molecule of Th2 cells, CRTH2.

    PubMed

    Schuligoi, Rufina; Sedej, Miriam; Waldhoer, Maria; Vukoja, Anela; Sturm, Eva M; Lippe, Irmgard T; Peskar, Bernhard A; Heinemann, Akos

    2009-01-01

    The major mast cell product PGD2 is released during the allergic response and stimulates the chemotaxis of eosinophils, basophils, and Th2-type T lymphocytes. The chemoattractant receptor homologous molecule of Th2 cells (CRTH2) has been shown to mediate the chemotactic effect of PGD2. PGH2 is the common precursor of all PGs and is produced by several cells that express cyclooxygenases. In this study, we show that PGH2 selectively stimulates human peripheral blood eosinophils and basophils but not neutrophils, and this effect is prevented by the CRTH2 receptor antagonist (+)-3-[[(4-fluorophenyl)sulfonyl] methyl amino]-1,2,3,4-tetrahydro-9H-carbazole-9-acetic acid (Cay10471) but not by the hematopoietic PGD synthase inhibitor 4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine (HQL79). In chemotaxis assays, eosinophils showed a pronounced migratory response toward PGH2, but eosinophil degranulation was inhibited by PGH2. Moreover, collagen-induced platelet aggregation was inhibited by PGH2 in platelet-rich plasma, which was abrogated in the presence of the D-type prostanoid (DP) receptor antagonist 3-[(2-cyclohexyl-2-hydroxyethyl)amino]-2,5-dioxo-1-(phenylmethyl)-4-imidazolidine-heptanoic acid (BWA868c). Each of these effects of PGH2 was enhanced in the presence of plasma and/or albumin. In eosinophils, PGH2-induced calcium ion (Ca2+) flux was subject to homologous desensitization with PGD2. Human embryo kidney (HEK)293 cells transfected with human CRTH2 or DP likewise responded with Ca2+ flux, and untransfected HEK293 cells showed no response. These data indicate that PGH2 causes activation of the PGD2 receptors CRTH2 and DP via a dual mechanism: by interacting directly with the receptors and/or by giving rise to PGD2 after catalytic conversion by plasma proteins.

  12. von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

    PubMed Central

    Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

    2007-01-01

    HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

  13. Prognostic impact of Beclin 1, p62/sequestosome 1 and LC3 protein expression in colon carcinomas from patients receiving 5-fluorouracil as adjuvant chemotherapy.

    PubMed

    Park, Jae Myung; Huang, Shengbing; Wu, Tsung-Teh; Foster, Nathan R; Sinicrope, Frank A

    2013-02-01

    Autophagy is a cellular degradation process that can be activated in tumor cells to confer stress tolerance. During autophagy initiation and autophagosome formation, Beclin 1 binds microtubule-associated protein-1 light chain 3 (LC3I) that is converted to its membrane-bound form (LC3II) and interacts with the ubiquitin-binding protein p62/sequestosome 1 (SQSTM1). We determined the association of Beclin 1, LC3 and p62 protein expression with clinical outcome in resected stage II and III colon carcinomas (n = 178) from participants in 5-fluororuacil (5-FU)-based adjuvant therapy trials. The immunopercentage for each marker was determined and dichotomized for analysis with overall survival (OS) using Cox models. We found that autophagy markers localized to the tumor cell cytoplasm and showed increased expression relative to normal epithelial cells. Overexpression of Beclin 1, LC3 and p62 proteins were detected in 69%, 79% and 85% of tumors, respectively. Expression levels were not significantly associated with clinicopathological variables. In a multivariable analysis adjusting for tumor grade, stage and patient age, Beclin 1 overexpression was independently associated with worse OS [hazard ratio (HR), 1.82; 95% confidence interval (CI), 1.0-3.3; p = 0.042] in patients who received 5-FU-based adjuvant therapy. Neither LC3 nor p62 overexpression was prognostic. In conclusion, Beclin 1 overexpression was associated with reduced survival in colon cancer patients treated with adjuvant 5-FU. These data are consistent with preclinical evidence indicating that autophagy can protect colon cancer cells from 5-FU and support the targeting of autophagy for therapeutic advantage in this malignancy.

  14. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    PubMed Central

    Kim, Seong K.; Kim, Seongman; Dai, Gan; Zhang, Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

    2012-01-01

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1,487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. PMID:21794889

  15. Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1.

    PubMed

    Zhang, Xiujun; Qu, Yinbo; Qin, Yuqi

    2016-01-01

    Heterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum ) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromatin status. Among the genes that exhibited significant differences between the hepA deletion strain (Δ hepA ) and the wild type (WT), most (95.0 %) were upregulated in Δ hepA compared with WT. The expression of the key transcription factor for cellulolytic enzyme gene (e.g., repressor CreA and activator ClrB) increased significantly. However, the deletion of hepA led to downregulation of prominent extracellular cellulolytic enzyme genes. Among the top 10 extracellular glycoside hydrolases (Amy15A, Amy13A, Cel7A/CBHI, Cel61A, Chi18A, Cel3A/BGLI, Xyn10A, Cel7B/EGI, Cel5B/EGII, and Cel6A/CBHII), in which secretion amount is from the highest to the tenth in P . oxalicum secretome, eight genes, including two amylase genes ( amy15A and amy13A ), all five cellulase genes ( cel7A / cbh1 , cel6A / cbh2 , cel7B / eg1 , cel5B / eg2 , and cel3A / bgl1 ), and the cellulose-active LPMO gene ( cel61A ) expression were downregulated. Results of chromatin accessibility real-time PCR (CHART-PCR) showed that the chromatin of all three tested upstream regions opened specifically because of the deletion of hepA in the case of two prominent cellulase genes cel7A/cbh1 and cel7B/eg1 . However, the open chromatin status did not occur along with the activation of cellulolytic enzyme gene expression. The overexpression of hepA upregulated the cellulolytic enzyme gene expression without chromatin modification. The overexpression of hepA remarkably activated the cellulolytic enzyme synthesis, not only in WT (~150

  16. Pseudomonas Pyocyanin Increases Interleukin-8 Expression by Human Airway Epithelial Cells

    PubMed Central

    Denning, Gerene M.; Wollenweber, Laura A.; Railsback, Michelle A.; Cox, Charles D.; Stoll, Lynn L.; Britigan, Bradley E.

    1998-01-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1α. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease. PMID:9826354

  17. [Dracorhodin perchlorate inhibit high glucose induce serum and glucocorticoid induced protein kinase 1 and fibronectin expression in human mesangial cells].

    PubMed

    Xie, Yifeng; Wang, Quansheng; Liu, Jianguo; Xie, Jiwen; Xue, Kaming; Tang, Qing

    2010-08-01

    To investigate the effect of dracorhodin perchlorate (DP) on inhibiting high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin (FN) expression in human mesangial cells (HMC), and its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN) . The HMC were divided into normal glucose group (NG group, 5.5 mmol x L(-1) D-glucose), normal glucose +low DP group (NG + LDP group, 5.5 mmol x L(-1) D-glucose +7.5 micromol x L(-1) DP), normal glucose +high DP group (NG + HDP group, 5.5 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP), high glucose group (HG group,25 mmol x L(-1) D-glucose), high glucose +low DP group (HG + LDP group, 25 mmol x L(-1) D-glucose + 7.5 micromol x L(-1) DP)and high glucose +high DP group (HG +HDP group, 25 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP). Each group was examined at 24 hours. The levels of SGK1 and FN mRNA was detected by real-time fluorescence quantitative PCR,and the expression of SGK1 and FN protein was detected by Western blot or indirect immunofluorescence. A basal level of SGK1 and FN in HMC were detected in NG group, and the level of SGK1 and FN mRNA and protein were not evidently different compared to that of NG group adding 7.5 micromol x L(-1) DP for 24 hours. On the other hand, the levels of SGK1 and FN mRNA and protein were obviously decreased by adding 15 micromol x L(-1) DP for 24 hours. Compared to NG group, the levels of SGK1 and FN mRNA and protein were increased in HG group after stimulating for 24 hours (P < 0.01). Compared to HG group, the level of SGK1 and FN mRNA and protein were evidently reduced in HG + LDP and HG + HDP groups by adding 7.5 micromol x L(-1) DP and 15 micromol x L(-1) DP for 24 hours (P < 0.01). Dracorhodin perchlorate can inhibit high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin(FN) expression in human mesangial cells, and this may be part of the mechanism of preventing and treating

  18. Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein.

    PubMed

    Okutani, R; Itoh, Y; Yamada, T; Yamaguchi, T; Singh, G; Yagisawa, H; Kawai, T

    1996-09-01

    Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity. We have expressed this protein in E. coli and characterized its physiochemical and biological properties. Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E. coli culture. The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus. On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1. The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical. Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities. These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1. We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.

  19. Expression of small heat shock proteins from pea seedlings under gravity altered conditions

    NASA Astrophysics Data System (ADS)

    Talalaev, Alexandr S.

    2005-08-01

    A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

  20. MSH-2 and MLH-1 Protein Expression in Muir Torre Syndrome-Related and Sporadic Sebaceous Neoplasms

    PubMed Central

    Morales-Burgos, Adisbeth; Sánchez, Jorge L.; Figueroa, Luz D.; De Jesús-Monge, Wilfredo E.; Cruz-Correa, Marcia R.; González-Keelan, Carmen; Nazario, Cruz María

    2009-01-01

    Background Muir-Torre Syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous neoplasm and internal malignancies. MTS-associated sebaceous neoplasms reveal mutations in DNA mismatch repair (MMR) genes and microsatellite instability. A significant part of MTS patients represents a phenotypic variant, the hereditary nonpolyposis colorectal cancer (HNPCC). A strong correlation between microsatellite instability and immunostaining has been demonstrated. The early recognition of sebaceous neoplasm as part of MTS, and their differentiation from sporadic sebaceous neoplasm may have an important application in a clinical setting. The absence of MLH-1 or MSH-2 expression by immunostaining identifies tumors with mismatch repair deficiency. Objectives Our aim is to determine whether an immunohistochemical approach, targeting DNA repair proteins MSH-2 and MLH-1 in MTS-related sebaceous neoplasm and their sporadic counterparts, can be used for their identification. Methods We examined 15 sebaceous neoplasms (including 6 internal malignancy- associated sebaceous neoplasms and 8 sporadic sebaceous neoplasms) from 11 patients for the expression of MSH-2 and MLH-1 by immunohistochemistry. Results Four of 5 internal malignancy-associated sebaceous neoplasms showed loss of expression of MSH-2 or MLH-1. Correlation of the immunostaining pattern of the sebaceous neoplasms and the patients’ positive history of colon carcinoma was 80%. Seven of 8 sporadic sebaceous neoplasms showed a positive expression of MSH-2 and MLH-1. The prevalence for loss of expression of MMR proteins in sebaceous neoplasms was 38.5%. MMR immunostaining had 87.5% specificity and 80% sensitivity. Limitations This study is limited by a small sample size, and by bias selection due to the use of non nationwide data-base as the resource of cases. Conclusions Our findings demonstrate that immunohistochemical testing for internal malignancy-associated sebaceous

  1. Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy.

    PubMed

    Gholamnezhadjafari, Reza; Tajik, Nader; Falak, Reza; Aflatoonian, Reza; Dehghan, Sanaz; Rezaei, Abbas

    2017-07-01

    Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.

  2. mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

    PubMed

    Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

    2014-12-01

    Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial

  3. SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

    PubMed

    Stephen, Tom L; Payne, Kyle K; Chaurio, Ricardo A; Allegrezza, Michael J; Zhu, Hengrui; Perez-Sanz, Jairo; Perales-Puchalt, Alfredo; Nguyen, Jenny M; Vara-Ailor, Ana E; Eruslanov, Evgeniy B; Borowsky, Mark E; Zhang, Rugang; Laufer, Terri M; Conejo-Garcia, Jose R

    2017-01-17

    Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Developmentally regulated expression of APG-1, a member of heat shock protein 110 family in murine male germ cells.

    PubMed

    Kaneko, Y; Kimura, T; Nishiyama, H; Noda, Y; Fujita, J

    1997-04-07

    Apg-1 encodes a heat shock protein belonging to the heat shock protein 110 family, and is inducible by a 32 degrees C to 39 degrees C heat shock. Northern blot analysis of the testis from immature and adult mice, and of the purified germ cells revealed the quantitative change of the apg-1 transcripts during germ cell development. By in situ hybridization histochemistry the expressions of the apg-1 transcripts were detected in germ cells at specific stages of development including spermatocytes and spermatids. Although heat-induction of the apg-1 transcripts was observed in W/Wv mutant testis lacking germ cells, it was not detected in wild-type testis nor in the purified germ cells. Thus, the apg-1 expression is not heat-regulated but developmentally regulated in germ cells, suggesting that APG-1 plays a role in normal development of germ cells.

  5. Apical Na(+)-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

    USDA-ARS?s Scientific Manuscript database

    Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus a...

  6. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    PubMed

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE-/- mice.

    PubMed

    Ganguly, Rituparna; Sahu, Soumyadip; Ohanyan, Vahagn; Haney, Rebecca; Chavez, Ronaldo J; Shah, Shivani; Yalamanchili, Siri; Raman, Priya

    2017-03-27

    Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE -/- ) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE -/- mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1 -/- /ApoE -/- double knockout mice were compared with age-matched hyperglycemic ApoE -/- littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE -/- mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

  8. Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1).

    PubMed

    Endres, Marcel; Kneitz, Susanne; Orth, Martin F; Perera, Ruwan K; Zernecke, Alma; Butt, Elke

    2016-09-27

    The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines.By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines.In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown.Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression.The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.

  9. Thyroid hormone activates rat liver adenosine 5,-monophosphate-activated protein kinase: relation to CaMKKb, TAK1 and LKB1 expression and energy status.

    PubMed

    Vargas, R; Ortega, Y; Bozo, V; Andrade, M; Minuzzi, G; Cornejo, P; Fernandez, V; Videla, L A

    2013-01-01

    AMP-activated protein kinase (AMPK) is a sensor of energy status supporting cellular energy homeostasis that may represent the metabolic basis for 3,3,,5-triiodo-L-thyronine (T3) liver preconditioning. Functionally transient hyperthyroid state induced by T3 (single dose of 0.1 mg/kg) in fed rats led to upregulation of mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of hepatic AMPK at 8 to 36 h after treatment. AMPK Thr 172 phosphorylation induced by T3 is associated with enhanced mRNA expression of the upstream kinases Ca2+ -calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) and transforming growth-factor-beta-activated kinase-1 (TAK1), with increased protein levels of CaMKKbeta and higher TAK1 phosphorylation, without changes in those of the liver kinase B1 (LKB1) signaling pathway. Liver contents of AMP and ADP were augmented by 291 percent and 44 percent by T3 compared to control values (p less than 0.05), respectively, whereas those of ATP decreased by 64% (p less than 0.05), with no significant changes in the total content of adenine nucleotides (AMP + ADP + ATP) at 24 h after T3 administration. Consequently, hepatic ATP/ADP content ratios exhibited 64 percent diminution (p less than 0.05) and those of AMP/ATP increased by 425 percent (p less than 0.05) in T3-treated rats over controls. It is concluded that in vivoT3 administration triggers liver AMPK upregulation in association with significant enhancements in AMPK mRNA expression, AMPK phosphorylation coupled to CaMKKbeta and TAK1 activation, and in AMP/ATP ratios, which may promote enhanced AMPK activity to support T3-induced energy consuming processes such as those of liver preconditioning.

  10. Targeted Proteomics-Driven Computational Modeling of Macrophage S1P Chemosensing*

    PubMed Central

    Manes, Nathan P.; Angermann, Bastian R.; Koppenol-Raab, Marijke; An, Eunkyung; Sjoelund, Virginie H.; Sun, Jing; Ishii, Masaru; Germain, Ronald N.; Meier-Schellersheim, Martin; Nita-Lazar, Aleksandra

    2015-01-01

    Osteoclasts are monocyte-derived multinuclear cells that directly attach to and resorb bone. Sphingosine-1-phosphate (S1P)1 regulates bone resorption by functioning as both a chemoattractant and chemorepellent of osteoclast precursors through two G-protein coupled receptors that antagonize each other in an S1P-concentration-dependent manner. To quantitatively explore the behavior of this chemosensing pathway, we applied targeted proteomics, transcriptomics, and rule-based pathway modeling using the Simmune toolset. RAW264.7 cells (a mouse monocyte/macrophage cell line) were used as model osteoclast precursors, RNA-seq was used to identify expressed target proteins, and selected reaction monitoring (SRM) mass spectrometry using internal peptide standards was used to perform absolute abundance measurements of pathway proteins. The resulting transcript and protein abundance values were strongly correlated. Measured protein abundance values, used as simulation input parameters, led to in silico pathway behavior matching in vitro measurements. Moreover, once model parameters were established, even simulated responses toward stimuli that were not used for parameterization were consistent with experimental findings. These findings demonstrate the feasibility and value of combining targeted mass spectrometry with pathway modeling for advancing biological insight. PMID:26199343

  11. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression.

    PubMed

    Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

    2006-08-08

    The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (P(gpdh-1)::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using P(gpdh-1)::GFP expression as a phenotype, we screened approximately 16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.

  12. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

    PubMed Central

    Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

    2006-01-01

    The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function. PMID:16880390

  13. Expression of Estrogen Receptor Coactivator Proline-, Glutamic Acid- and Leucine-Rich Protein 1 within Paraspinal Muscles in Adolescents with Idiopathic Scoliosis

    PubMed Central

    Skibinska, Izabela; Tomaszewski, Marek; Andrusiewicz, Miroslaw; Urbaniak, Paulina; Czarnecka-Klos, Roza; Shadi, Milud; Kotwicki, Tomasz; Kotwicka, Malgorzata

    2016-01-01

    Purpose The aim of this study was to detect and assess the estrogen receptor (ESR) coactivator PELP1 expression within human paraspinal skeletal muscles in patients suffering from idiopathic scoliosis. Methods During surgical correction of scoliosis the muscle biopsies harvested in 29 females. Presence of PELP1, ESR1 and ESR2 genes transcripts was studied using RT-qPCR technique while immunohistochemistry and western blot methods were used to detect the PEPL1 protein presence. Results PELP1 expression in deep paraspinal muscles revealed higher than in superficial back muscles (p = 0.005). Positive immunohistochemical staining for PELP1 was observed in the nuclei of the paraspinal muscle cells. Western blot revealed PELP1 protein in all samples. No significant difference in PELP1 expression between the convex and the concave scoliosis side (p>0.05) was found. In deep paraspinal back muscles, a significant correlation between the PELP1 expression level on the concave side and the Cobb angle (r = 0.4; p<0.05) was noted as well as between the PELP1 and ESR1 expression level (r = 0.7; p<0.05) while no correlation between PELP1 and ESR2 expression level was found. Conclusion To our knowledge, three techniques for the first time demonstrated the presence of the PELP1 in paraspinal muscles of patients with idiopathic scoliosis. The PELP1 potential regulatory impact on back muscle function is to be further investigated. PMID:27045366

  14. Lack of Correlation between Aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 Protein Expression and Promoter Methylation in Squamous Cell Carcinoma Accompanying Candida albicans-Induced Inflammation

    PubMed Central

    Terayama, Yui; Matsuura, Tetsuro; Ozaki, Kiyokazu

    2016-01-01

    Hyperplastic candidiasis is characterized by thickening of the mucosal epithelia with Candida albicans infection with occasional progression to squamous cell carcinoma (SCC). C. albicans is a critical factor in tumor development; however, the oncogenic mechanism is unclear. We have previously produced an animal model for hyperplastic candidiasis in the rat forestomach. In the present study, we investigate whether impaired DNA methylation and associated protein expression of tumor suppressor and DNA repair genes are involved in the SCC carcinogenesis process using this hyperplastic candidiasis model. Promoter methylation and protein expression were analyzed by methylation specific PCR and immunohistochemical staining, respectively, of 5 areas in the forestomachs of alloxan-induced diabetic rats with hyperplastic candidiasis: normal squamous epithelia, squamous hyperplasia, squamous hyperplasia adjacent to SCC, squamous hyperplasia transitioning to SCC, and SCC. We observed nuclear p16 overexpression despite increases in p16 gene promoter methylation during the carcinogenic process. TIMP3 and RAR-β2 promoter methylation progressed until the precancerous stage but disappeared upon malignant transformation. In comparison, TIMP3 protein expression was suppressed during carcinogenesis and RAR-β2 expression was attenuated in the cytoplasm but enhanced in nuclei. ERCC1 and BRCA1 promoters were not methylated at any stage; however, their protein expression disappeared beginning at hyperplasia and nuclear protein re-expression in SCC was observed only for ERCC1. These results suggest that aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 expression might occur that is inconsistent with the respective gene promoter methylation status, and that this overexpression might serve to promote the inflammatory carcinogenesis caused by C. albicans infection. PMID:27410681

  15. Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli.

    PubMed

    Kataeva, Irina; Chang, Jessie; Xu, Hao; Luan, Chi-Hao; Zhou, Jizhong; Uversky, Vladimir N; Lin, Dawei; Horanyi, Peter; Liu, Z J; Ljungdahl, Lars G; Rose, John; Luo, Ming; Wang, Bi-Cheng

    2005-01-01

    Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is

  16. Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii.

    PubMed

    Lee, Song F; Li, Yi-Jing; Halperin, Scott A

    2009-11-01

    One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

  17. Rhythmic expression of DEC2 protein in vitro and in vivo.

    PubMed

    Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

    2016-06-01

    Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

  18. Triptorelin and cetrorelix induce immune responses and affect uterine development and expressions of genes and proteins of ESR1, LHR, and FSHR of mice.

    PubMed

    Wei, Suocheng; Guo, Huiling; Gong, Zhuandi; Zhang, Fengwei; Ma, Zhongren

    2016-06-01

    GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus. The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri. One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n = 15) were subcutaneously injected with 10, 20, and 40 μg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40 μg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins. Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p < 0.05), respectively. Uterine weights of TRI-2, TRI-3 reduced by 28.57% and 11.43% (p < 0.05), respectively. The uterine cavity in CET-1, CET-2, and CET-3 increased; the uterine wall became thick. The cytoplasm of endometrial epithelial cells (EEC) increased slightly. In TRI group, the uterine wall thinned. Uterine cavity became narrow slightly in TRI-1. Numbers of uterine glands reduced. The endometrium epithelial thickness (EET) in CET-1 and CET-2 increased by 68.21% and 79.46% (p < 0.05), respectively. EET in TRI-1 was decreased by 13.69%. Uterine wall thicknesses (UWT) in CET-1 and CET-2 were higher than CG, with the increment of 28.59% and 30.72%. UWT of TRI-1, TRI-2, and TRI-3 reduced by 29.35, 15.36, and 14.41%, respectively. Expressions of ESR1, FSHR, and LHR mRNAs in CET and TRI mice increased. ESR1 and FSHR protein levels increased in all

  19. Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12

    PubMed Central

    Tan, Jiangning; Tedrow, John R.; Nouraie, Mehdi; Dutta, Justin A.; Miller, David T.; Li, Xiaoyun; Yu, Shibing; Chu, Yanxia; Juan-Guardela, Brenda; Kaminski, Naftali; Ramani, Kritika; Biswas, Partha S.; Zhang, Yingze

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow–derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury. PMID:28179498

  20. Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

    PubMed Central

    Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

    2006-01-01

    Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267