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Sample records for factor egf conjugated

  1. Preparation of epidermal growth factor (EGF) conjugated iron oxide nanoparticles and their internalization into colon cancer cells

    International Nuclear Information System (INIS)

    Creixell, Mar; Herrera, Adriana P.; Ayala, Vanessa; Latorre-Esteves, Magda; Perez-Torres, Marianela; Torres-Lugo, Madeline; Rinaldi, Carlos

    2010-01-01

    Epidermal growth factor (EGF) was conjugated with carboxymethyldextran (CMDx) coated iron oxide magnetic nanoparticles using carbodiimide chemistry to obtain magnetic nanoparticles that target the epidermal growth factor receptor (EGFR). Epidermal growth factor modified magnetic nanoparticles were colloidally stable when suspended in biological buffers such as PBS and cell culture media. Both targeted and non-targeted nanoparticles were incubated with CaCo-2 cancer cells, known to overexpress EGFR. Nanoparticle localization within the cell was visualized by confocal laser scanning microscopy and light microscopy using Prussian blue stain. Results showed that targeted magnetic nanoparticles were rapidly accumulated in both flask-shaped small vesicles and large circular endocytic structures. Internalization patterns suggest that both clathrin-dependent and clathrin-independent receptors mediated endocytosis mechanisms are responsible for nanoparticle internalization.

  2. Epidermal growth factor (EGF) as a potential targeting agent for delivery of boron to malignant gliomas

    International Nuclear Information System (INIS)

    Capala, J.; Barth, R.F.; Adams, D.M.; Bailey, M.Q.; Soloway, A.H.; Carlsson, J.

    1994-01-01

    The majority of high grade gliomas express an amplified epidermal growth factor receptor (EGFR) gene, and this often is associated with an increase in cell surface receptor expression. The rapid internalization and degradation of EGF-EGFR complexes, as well as their high affinity make EGF a potential targeting agent for delivery of 10 B to tumor cells with an amplified number of EGFR. Human glioma cells can expresses as many as 10 5 -10 6 EGF receptors per cell, and if these could be saturated with boronated EGF, then > 10 8 boron atoms would be delivered per cell. Since EGF has a comparatively low molecular weight (∼ 6 kD), this has allowed us to construct relatively small bioconjugates containing ∼ 900 boron atoms per EGF molecule 3 , which also had high affinity for EGFR on tumor cells. In the present study, the feasibility of using EGF receptors as a potential target for therapy of gliomas was investigated by in vivo scintigraphic studies using 131 I- or 99m T c -labeled EGF in a rat brain tumor model. Our results indicate that intratumorally delivered boron- EGF conjugates might be useful for targeting EGFR on glioma cells if the boron containing moiety of the conjugates persisted intracellularly. Further studies are required, however, to determine if this approach can be used for BNCT of the rat glioma

  3. Heparin-Binding EGF-like Growth Factor (HB-EGF) Therapy for Intestinal Injury: Application and Future Prospects

    Science.gov (United States)

    Yang, Jixin; Su, Yanwei; Zhou, Yu; Besner, Gail E.

    2014-01-01

    Throughout the past 20 years, we have been investigating the potential therapeutic roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor family, in various models of intestinal injury including necrotizing enterocolitis (NEC), intestinal ischemia/reperfusion (I/R) injury, and hemorrhagic shock and resuscitation (HS/R). Our studies have demonstrated that HB-EGF acts as an effective mitogen, a restitution-inducing reagent, a cellular trophic factor, an anti-apoptotic protein and a vasodilator, via its effects on various cell types in the intestine. In the current paper, we have reviewed the application and therapeutic effects of HB-EGF in three classic animal models of intestinal injury, with particular emphasis on its protection of the intestines from NEC. Additionally, we have summarized the protective functions of HB-EGF on various target cells in the intestine. Lastly, we have provided a brief discussion focusing on the future development of HB-EGF clinical applications for the treatment of various forms of intestinal injury including NEC. PMID:24345808

  4. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    Energy Technology Data Exchange (ETDEWEB)

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi; Sekine, Yoshimoto [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Yamada, Kazuo [Laboratory of Molecular Psychiatry, RIKEN Brain Science Institute, Saitama (Japan); Tsujii, Masatsugu [Faculty of Sociology, Chukyo University, Toyota, Aichi (Japan); [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Yoshikawa, Takeo [Laboratory of Molecular Psychiatry, RIKEN Brain Science Institute, Saitama (Japan); Miyachi, Taishi; Tsuchiya, Kenji; Sugihara, Gen-ichi; Matsuzaki, Hideo [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); Iwata, Yasuhide; Suzuki, Katsuaki [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Mori, Norio [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Graduate School of Medicine, Osaka University (Japan); Ouchi, Yasuomi [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); [The Positron Medical Center, Hamamatsu Medical Center, Hamamatsu (Japan); Sugiyama, Toshiro [Aichi Children' s Health and Medical Center, Obu, Aichi (Japan); Takei, Nori [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan)

    2007-09-07

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.

  5. SNP analyses of growth factor genes EGF, TGFβ-1, and HGF reveal haplotypic association of EGF with autism

    International Nuclear Information System (INIS)

    Toyoda, Takao; Nakamura, Kazuhiko; Yamada, Kazuo; Thanseem, Ismail; Anitha, Ayyappan; Suda, Shiro; Tsujii, Masatsugu; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Miyachi, Taishi; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Kawai, Masayoshi; Sekine, Yoshimoto; Tsuchiya, Kenji; Sugihara, Gen-ichi; Ouchi, Yasuomi; Sugiyama, Toshiro; Takei, Nori; Yoshikawa, Takeo; Mori, Norio

    2007-01-01

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-β (TGFβ) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGFβ1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGFβ1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism

  6. Heparin-Binding EGF-like Growth Factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas

    Science.gov (United States)

    Todd, Levi; Volkov, Leo I.; Zelinka, Chris; Squires, Natalie; Fischer, Andy J.

    2015-01-01

    Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, Heparin-Binding EGF-like Growth Factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF stimulated not only MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas. PMID:26500021

  7. Brief study about the distribution of recombinant human Epidermic Growth Factor (rh-EGF)

    International Nuclear Information System (INIS)

    Rodriguez Garcia, J.C.; De Dios D Espaux, R.; Bello Garciga, J.L.

    1997-01-01

    This report describes results of the study about biodistribution of I-125 recombinant human Epidermic Growth Factor (rhEGF). The radiolabelled product was administrated to Sprague Dawley rats in three different ways: intramuscular, subcutaneous and epidermic; the highest concentration of EGF in blood was found 4 hours after rhEGF administration, with a greater distribution in the plasma with regard to cellular pellet. The slowest plasma clearance corresponded to the intramuscular administration. The highest concentration of radiolabelled rhEGF was found in liver, kidney and intestine. It was found that radiolabelled EGF is excreted mainly throughout urine and faeces although other excretion pathways could exist

  8. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  9. Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

    Science.gov (United States)

    Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

    2017-04-01

    The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Epidermal growth factor (EGF) inhibits stimulated thyroid hormone secretion in the mouse

    International Nuclear Information System (INIS)

    Ahren, B.

    1987-01-01

    It is known that epidermal growth factor (EGF) inhibits iodide uptake in the thyroid follicular cells and lowers plasma levels of thyroid hormones upon infusion into sheep and ewes. In this study, the effects of EGF on basal and stimulated thyroid hormone secretion were investigated in the mouse. Mice were pretreated with 125 I and thyroxine; the subsequent release of 125 I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was not altered by intravenous injection of EGF (5 micrograms/animal). However, the radioiodine secretion stimulated by both TSH (120 microU/animal) and vasoactive intestinal peptide (VIP; 5 micrograms/animal) were inhibited by EGF (5 micrograms/animal). At a lower dose level (0.5 microgram/animal), EGF had no influence on stimulated radioiodine secretion. In conclusion, EGF inhibits stimulated thyroid hormone secretion in the mouse

  11. The role of sialoadenectomy and epıdermal growth factor (EGF) in ...

    African Journals Online (AJOL)

    USER

    2010-05-17

    May 17, 2010 ... result, epidermal growth factor was concluded to have an important role in skin development. Key words: Epidermal growth factor, ... sialoadenectomy on epiderm and the role of EGF and antiserum EGF in prevention of .... remarkable finding in skin healing of sialoadenectomy and normal rats. According to ...

  12. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

    Science.gov (United States)

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk

    2017-01-01

    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  13. The effects of chronic administration of epidermal growth factor (EGF) to rats on the levels of endogenous EGF in the submandibular glands and kidneys

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Jøgensen, P E; Poulsen, Steen Seier

    1996-01-01

    Epidermal growth factor (EGF) is mainly produced in the submandibular glands (SMG) and in the kidneys. It has recently been reported that EGF-related ligands may induce their own biosynthesis (autoinduction) in vitro. In the present paper, we investigated whether chronic systemic treatment with E...

  14. Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure.

    Science.gov (United States)

    Schaudies, R P; Nonclercq, D; Nelson, L; Toubeau, G; Zanen, J; Heuson-Stiennon, J A; Laurent, G

    1993-09-01

    The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic

  15. The preventive effect of recombinant human growth factor (rhEGF) on the recurrence of radiodermatitis

    International Nuclear Information System (INIS)

    Ryu, Seung-Hee; Kim, Yeun-Hwa; Lee, Sang-Wook; Hong, Joon-Pio

    2010-01-01

    The effects of topical application of recombinant human epidermal growth factor (rhEGF) on wound healing and the recurrence of radiodermatitis were assessed in the irradiated skin of BALB/c Nu/Nu mice. Mice irradiated with 45 Gy of radiation were divided into 5 groups and treated with 10, 50, and 100 μg/g rhEGF ointment, vehicle alone, or no treatment (control) for 6 months. Wounds were observed initially in all groups and complete healing time (HT 100 ) for initial wound repair did not differ significantly among groups. However, the rate of recurrence over 6 months was significantly lower in the EGF-treated groups than in the control group (p<0.05). Histological examination showed that treatment with the optimum dose of EGF (50 μg/g) accelerated normal wound healing when compared with the higher dose of EGF (100 μg/g), vehicle alone, or no treatment, with the latter group showing irregular epidermal thickness, poor definition of epidermis and dermis, and unstable dermal structure. Collagen distribution was also significantly increased in mice treated with 50 μg/g rhEGF (p<0.05) compared with the control or vehicle-treated group. Taken together, these results indicate that treatment with exogenous EGF (50 μg/g dose) can enhance radiation-induced wound repair while preserving structural tissue stability and preventing the recurrence of radiodermatitis. (author)

  16. Ticagrelor Improves Endothelial Function by Decreasing Circulating Epidermal Growth Factor (EGF

    Directory of Open Access Journals (Sweden)

    Francesco Vieceli Dalla Sega

    2018-04-01

    Full Text Available Ticagrelor is one of the most powerful P2Y12 inhibitor. We have recently reported that, in patients with concomitant Stable Coronary Artery Disease (SCAD and Chronic Obstructive Pulmonary Disease (COPD undergoing percutaneous coronary intervention (PCI, treatment with ticagrelor, as compared to clopidogrel, is associated with an improvement of the endothelial function (Clinical Trial NCT02519608. In the present study, we showed that, in the same population, after 1 month treatment with ticagrelor, but not with clopidogrel, there is a decrease of the circulating levels of epidermal growth factor (EGF and that these changes in circulating levels of EGF correlate with on-treatment platelet reactivity. Furthermore, in human umbilical vein endothelial cells (HUVEC incubated with sera of the patients treated with ticagrelor, but not with clopidogrel there is an increase of p-eNOS levels. Finally, analyzing the changes in EGF and p-eNOS levels after treatment, we observed an inverse correlation between p-eNOS and EGF changes only in the ticagrelor group. Causality between EGF and eNOS activation was assessed in vitro in HUVEC where we showed that EGF decreases eNOS activity in a dose dependent manner. Taken together our data indicate that ticagrelor improves endothelial function by lowering circulating EGF that results in the activation of eNOS in the vascular endothelium.

  17. Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

    Science.gov (United States)

    Choi, Yohan; Wilson, Kalin; Hannon, Patrick R; Rosewell, Katherine L; Brännström, Mats; Akin, James W; Curry, Thomas E; Jo, Misung

    2017-06-01

    In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells. Copyright © 2017 Endocrine Society

  18. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy.

    Directory of Open Access Journals (Sweden)

    Catarina Oliveira Silva

    Full Text Available The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm showed a plasmon absorption band located within the near-infrared range (650-900 nm, optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%. Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.

  19. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    Science.gov (United States)

    Silva, Catarina Oliveira; Petersen, Steffen B.; Reis, Catarina Pinto; Rijo, Patrícia; Molpeceres, Jesús; Fernandes, Ana Sofia; Gonçalves, Odete; Gomes, Andreia C.; Correia, Isabel; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2016-01-01

    The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100–200 nm) showed a plasmon absorption band located within the near-infrared range (650–900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0–25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue. PMID:27788212

  20. Effects of recombinant human epidermal growth factor (rhEGF) on experimental radiation-induced oral mucositis in rats

    International Nuclear Information System (INIS)

    Jung, Kwon Il; Kim, Sun Hee; Moon, Soo Young; Kim, Yeon Wha; Hong, Joon Pio; Lee, Sang Wook; Kim, Hyun Sook

    2006-01-01

    Oral mucositis is a common toxicity of radiation or chemotherapy, which is used a treatment for head and neck cancer. We investigated effects of recombinant human epidermal growth factor (rhEGF) on radiation-induced oral mucositis in rat model. Spraque-Dawley rats (7 per group) exposed to a single dose of 25 Gy (day 0) on their head, except for one group, were randomly divided into un-treated, vehicle-treated, and two rhEGF-treated groups. Rats were topically applied with rhEGF (15 or 30 μ g/oral cavity/day) or vehicle to their oral mucosa. Survival rate of rats, weight changes, and food intakes were examined from day 0 to 18 after radiation. Histology study was performed from oral mucosa of rats at day 7 and 18 after radiation. rhEGF-treated groups (15 or 30 μ g/day) showed all survival rate 33%, whereas un-treated and vehicle-treated groups showed all survival rate 0% at the end of experiment. rhEGF-treated groups statistically had less weight loss compared to vehicle-treated group from day 2 to 7 after radiation. Food intake of rats with rhEGF treatment turned to increase at day 14 after radiation. At 7 day after radiation, un-treated and vehicle-treated groups showed severe pseudomembraneous of ulcerative oral mucositis. On the other hand, rhEGF-treated groups had no more than cellular swelling and degeneration of epidermal cells in oral mucosa of rats. These results suggest that rhEGF has significantly positive effects on radiation-induced oral mucositis in rats. rhEGF display a therapeutic potential on a clinical level

  1. Pharmacological inhibition of heparin-binding EGF-like growth factor promotes peritoneal angiogenesis in a peritoneal dialysis rat model.

    Science.gov (United States)

    Li, Zhenyuan; Yan, Hao; Yuan, Jiangzi; Cao, Liou; Lin, Aiwu; Dai, Huili; Ni, Zhaohui; Qian, Jiaqi; Fang, Wei

    2018-04-01

    Molecular mechanisms of peritoneal dialysis (PD) ultrafiltration failure, peritoneal neo-angiogenesis, and fibrosis remain to be determined. We aimed to determine the role of heparin-binding EGF-like growth factor (HB-EGF) inhibition on angiogenesis of peritoneal membrane in a PD rat model. 32 male Wistar rats were assigned into (1) control group; (2) uremic non-PD group: subtotal nephrectomy-induced uremic rats without PD; (3) uremic rats subjected to PD: uremic rats that were dialyzed with Dianeal ® for 4 weeks; (4) CRM 197 group: dialyzed uremic rats were supplemented with CRM197, a specific HB-EGF inhibitor. Peritoneal transport function was examined by peritoneal equilibration test. Expression of HB-EGF and EGFR in peritoneal samples were examined by real-time PCR, immunohistochemical staining, and western blot. Progressive angiogenesis and fibrosis were observed in uremic PD rats, and there were associated with decreased net ultrafiltration (nUF), increased permeability of peritoneal membrane, and reduced expression of HB-EGF and EGFR protein and mRNA in uremic PD rats compared to uremic non-PD or control groups (both p CRM197 significantly induced peritoneal membrane permeability, decreased nUF, increased higher vessel density, and reduced pericyte count compared to that of uremic PD rats. The levels of HB-EGF and EGFR expression negatively correlated with vessel density in peritoneal membrane (both p < 0.001). PD therapy was associated with peritoneal angiogenesis, functional deterioration, and downregulation of HB-EGF/EGFR. Pharmacological inhibition of HB-EGF promoted PD-induced peritoneal angiogenesis and fibrosis and ultrafiltration decline, suggesting that HB-EGF downregulation contributes to peritoneal functional deterioration in the uremic PD rat model.

  2. Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise G; Nielsen, Jens N; Ornskov, Dorthe

    2007-01-01

    Introduction. EGF/EGFR interactions are important mechanisms behind colorectal tumour development and growth. Recently a single nucleotide polymorphism in the EGF gene has been identified (EGF A61G). It may be a potential predictor for survival of patients receiving EGFR-inhibitor cetuximab...

  3. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Takayo; Yoshida, Yuichi [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan); Imai, Yasuharu [Department of Gastroenterology, Ikeda Municipal Hospital, Ikeda, Osaka (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University, Graduate School of Medicine and Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center (ProMRes), Ehime University, Shitsukawa, Toon, Ehime (Japan); Iwamoto, Ryo; Mekada, Eisuke [Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Takehara, Tetsuo [Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka (Japan)

    2013-07-26

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

  4. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    International Nuclear Information System (INIS)

    Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi; Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro; Imai, Yasuharu; Higashiyama, Shigeki; Iwamoto, Ryo; Mekada, Eisuke; Takehara, Tetsuo

    2013-01-01

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis

  5. Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2.

    OpenAIRE

    Hayase, N; Ishiyama, A; Niwano, M

    1994-01-01

    We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted E...

  6. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

    Science.gov (United States)

    Long, D.L.; Ulici, V.; Chubinskaya, S.; Loeser, R.F.

    2015-01-01

    Objective We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. Methods HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and OA human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. Results HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. Conclusion HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. PMID:25937027

  7. Stimulation of chondrocytes in vitro by gene transfer with plasmids coding for epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF)

    DEFF Research Database (Denmark)

    Schmal, H; Mehlhorn, A T; Zwingmann, J

    2005-01-01

    Human epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF) influence critical characteristics of chondrocytes. The effects on metabolism and differentiation were evaluated following transfection using specific plasmids coding for both cytokines. Chondrocytes were isolated from...... of recombinant hEGF and bFGF resulted in a significant increase in cell proliferation and glucosaminoglycan production. Chondrocytes were transfected with vectors coding for either hEGF or bFGF and the production of these proteins was measured in supernatants by ELISA. Expression kinetics showed different...... patterns: hEGF was detectable 2.5 days following transfection and peaked at day 5.5, whereas bFGF-production reached its maximum 1.5 days after transfection, declining thereafter. Chondrocytes endogenously produced significant amounts of bFGF within 5 days following isolation. Proliferation of h...

  8. Serum Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) as a Biomarker for Primary Ovarian Cancer.

    Science.gov (United States)

    Miyata, Kohei; Yotsumoto, Fusanori; Fukagawa, Satoshi; Kiyoshima, Chihiro; Ouk, Nam Sung; Urushiyama, Daichi; Ito, Tomohiro; Katsuda, Takahiro; Kurakazu, Masamitsu; Araki, Ryota; Sanui, Ayako; Miyahara, Daisuke; Murata, Masaharu; Shirota, Kyoko; Yagi, Hiroshi; Takono, Tadao; Kato, Kiyoko; Yaegashi, Nobuo; Akazawa, Kohei; Kuroki, Masahide; Yasunaga, Shin'ichiro; Miyamoto, Shingo

    2017-07-01

    Ovarian cancer is the most lethal malignancy among gynaecological cancers. Although many anticancer agents have been developed for the treatment of ovarian cancer, it continues to have an extremely poor prognosis. Heparin-binding epidermal growth factor-like grown factor (HB-EGF) has been reported to be a rational therapeutic target for ovarian cancer. Here, we evaluated the clinical significance of serum HB-EGF by examining the association between prognosis and serum HB-EGF levels in patients with primary ovarian cancer. We found that high serum HB-EGF concentrations were significantly associated with poor prognosis in a combined cohort of patients with all stages of ovarian cancer, as well as in a subset of patients with advanced disease. In addition, serum HB-EGF levels increased as the cancer advanced. These data suggest that serum HB-EGF may be a target for the design of novel therapies for ovarian cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  9. Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells

    International Nuclear Information System (INIS)

    Long, G.J.; Rosen, J.F.

    1991-01-01

    EGF, a single chain polypeptide growth factor important for many cellular functions including glycolysis and protein phophorylation, is known to modulate calcium metabolism in several cell systems. It has been shown that EGF causes an increase in Ca 2+ influx and accumulation of inositol triphosphate, and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; hence, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if cell functions controlled by EGF were impaired. Cells were labelled with 45 Ca (1.87 mM Ca) for 20 hr in the presence of 5 μM Pb, 50 ng/ml EGF or μM Pb and 50 ng/ml EGF. Following an EGTA rinse, kinetic parameters were determined from 45 Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45 Ca. 5 μM Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast exchanging, quantitatively small calcium pools, S 1 and S 2 to the slow exchanging, quantitatively large S 2 . There was also a 50% increase in total cell calcium in cells treated with 5 μM Pb and 50 ng/ml EGF over cells treated with 50 ng/ml EGF alone. There was also a 25% decrease in the half-time for calcium exchange from S 3 to S 1 was also decreased. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis by EGF and may therefore perturb functions that are modulated by EGF via the calcium messenger system

  10. EFFECTS OF EPIDERMAL GROWTH FACTOR (EGF), TRANSFORMING GROWTH FACTOR- (TGF), AND 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON FUSION OF EMBRYONIC PALATES IN SERUM-FREE ORGAN CULTURE USING WILD-TYPE, EGF KNOCKOUT, AND TGF KNOCKOUT MOUSE STRAINS

    Science.gov (United States)

    Backround: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor- (TGF) in the palate and affects proliferation and different...

  11. Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

    Science.gov (United States)

    Huber, Birgit; Czaja, Alina Maria; Kluger, Petra Juliane

    2016-05-01

    The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used. © 2016 International Federation for Cell Biology.

  12. Study on the changes of serum levels of polypeptide growth factors (EGF, TGF-α) and related hormones (gastrin, somatostatin) in patients with peptic ulcer

    International Nuclear Information System (INIS)

    Cao Jianfan; Ma Yunbao; Zhang Xiaoyi

    2005-01-01

    Objective: To study the possible roles of EGF, TGF-α, gastrin and somatostatin in the pathogenesis of peptic ulcer by measuring the changes of serum levels of those four parameters in the patients with peptic ulcer. Methods: Serum levels of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), gastrin (Gas) and somatostatin (SS) were measured with RIA in 30 patients with gastric ulcer, 32 patients with duodenal ulcer and 30 controls. Results: Serum levels of gastrin and EGF were significantly higher in the patients with peptic ulcer than those in the controls (both P 0.05). However, serum TGF-α levels were significantly lower in the ulcer patients than those in the controls (P<0.01). Conclusion: Changes of serum levels of gastrin, EGF and TGF-α were quite significant in the ulcer patients and determining of which might be of clinical meanings. Determination of somatostatin changes seemed to be of less importance. (authors)

  13. The neuroprotective effects of milk fat globule-EGF factor 8 against oligomeric amyloid β toxicity

    Directory of Open Access Journals (Sweden)

    Li Endong

    2012-06-01

    Full Text Available Abstract Background Phosphatidylserine receptor is a key molecule that mediates the phagocytosis of apoptotic cells. Milk fat globule-EGF factor 8 (MFG-E8 is a phosphatidylserine receptor that is expressed on various macrophage lineage cells, including microglia in the central nervous system (CNS. Targeted clearance of degenerated neurons by microglia is essential to maintain healthy neural networks. We previously showed that the CX3C chemokine fractalkine is secreted from degenerated neurons and accelerates microglial clearance of neuronal debris via inducing the release of MFG-E8. However, the mechanisms by which microglia produce MFG-E8 and the precise functions of MFG-E8 are unknown. Methods The release of MFG-E8 from microglia treated with conditioned medium from neurons exposed to neurotoxic substances, glutamate or oligomeric amyloid β (oAβ was measured by ELISA. The neuroprotective effects of MFG-E8 and MFG-E8 − induced microglial phagocytosis of oAβ were assessed by immunocytochemistry. The effects of MFG-E8 on the production of the anti-oxidative enzyme hemeoxygenase-1 (HO-1 were determined by ELISA and immunocytochemisty. Results MFG-E8 was induced in microglia treated with conditioned medium from neurons that had been exposed to neurotoxicants, glutamate or oAβ. MFG-E8 significantly attenuated oAβ-induced neuronal cell death in a primary neuron − microglia coculture system. Microglial phagocytosis of oAβ was accelerated by MFG-E8 treatment due to increased CD47 expression in the absence of neurotoxic molecule production, such as tumor necrosis factor-α, nitric oxide, and glutamate. MFG-E8 − treated microglia induced nuclear factor E(2 − related factor 2 (Nrf2 − mediated HO-1 production, which also contributed to neuroprotection. Conclusions These results suggest that microglia release MFG-E8 in response to signals from degenerated neurons and that MFG-E8 protects oAβ-induced neuronal cell death

  14. Contribution of transcription factor, SP1, to the promotion of HB-EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma

    International Nuclear Information System (INIS)

    Miyata, Kohei; Yotsumoto, Fusanori; Nam, Sung Ouk; Odawara, Takashi; Manabe, Sadao; Ishikawa, Toyokazu; Itamochi, Hiroaki; Kigawa, Junzo; Takada, Shuji; Asahara, Hiroshi; Kuroki, Masahide; Miyamoto, Shingo

    2014-01-01

    Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′-deletion promoter constructs identified a GC-rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells

  15. Improvement of Atrophic Acne Scars in Skin of Color Using Topical Synthetic Epidermal Growth Factor (EGF) Serum: A Pilot Study.

    Science.gov (United States)

    Stoddard, Marie Alexia; Herrmann, Jennifer; Moy, Lauren; Moy, Ronald

    2017-04-01

    BACKGROUND: Atrophic scarring in skin of color is a common, permanent, and distressing result of uncontrolled acne vulgaris. Ablative lasers and chemical peels are frequently used to improve the appearance of atrophic scars, primarily through the stimulation of collagen and elastin; however, these treatment modalities are associated with risks, such as dyspigmentation and hypertrophic scarring, especially in patients with darker skin. OBJECTIVE: We evaluated the efficacy of topically applied synthetic epidermal growth factor (EGF) serum in reducing the appearance of atrophic acne scars in skin of color. METHODS: A single-center clinical trial was performed on twelve healthy men and women (average age 32.5) with Fitzpatrick Type IV-V skin and evidence of facial grade II-IV atrophic acne scars. Subjects applied topical EGF serum to the full-face twice daily for 12 weeks. Scar improvement was investigated at each visit using an Investigator Global Assessment (IGA), a Goodman grade, clinical photography, and patient self-assessment. RESULTS: Eleven subjects completed the trial. Compared to baseline, there was an improvement in mean IGA score from 3.36 (SEM = 0.15) to 2.18 (SEM = 0.33). Mean Goodman grade was reduced from 2.73 (SEM = 0.19) to 2.55 (SEM = 0.21). Of the eleven pairs of before and after photographs, nine were correctly chosen as the post-treatment image by a blind investigator. On self-assessment, 81% reported a "good" to "excellent" improvement in their scars compared to baseline (P = 0.004). CONCLUSION: Topical EGF may improve the appearance of atrophic acne scars in skin of color. Additional, larger studies should be conducted to better characterize improvement. J Drugs Dermatol. 2017;16(4):322-326..

  16. Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Krogh, T N; Støving, René Klinkby

    1997-01-01

    We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an aver......We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.......2% and an average recovery of 105% in serum and 98% in urine. Comparison of FA1 in amniotic fluid, serum and urine revealed parallel titration curves, identical elution volumes following size chromatography, immunological identity and similar profiles when analysed by MALDI-MS. The reference interval for serum FA1...... was 12.3-46.6 ng/ml and the levels were 10 times higher in patients with renal failure. FA1 showed no diurnal variation, no variation during the menstrual cycle and was not influenced by the acute phase reaction. In humans (n = 10) the renal clearance of FA1 was 11 ml/min and an identical high renal...

  17. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    International Nuclear Information System (INIS)

    Kosheverova, Vera V.; Kamentseva, Rimma S.; Gonchar, Ilya V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-01-01

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  18. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Gonchar, Ilya V., E-mail: ample@mail.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kharchenko, Marianna V., E-mail: mariannakharchenko@gmail.com [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kornilova, Elena S., E-mail: lenkor@mail.cytspb.rssi.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 29, Polytechnicheskaya, St.Petersburg, 195251 (Russian Federation)

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  19. Bg1II polymorphism of the epidermal growth factor receptor (EGF-R) gene

    Energy Technology Data Exchange (ETDEWEB)

    Biunno, I; Pozzi, M R; Radice, P; Mondini, P; Pierotti, M A; Porta, G D [Istituto Nazionale Tumori, Milan (Italy); Haley, J; Waterfield, M D [Ludwig Institute for Cancer Research, London (England)

    1988-08-11

    A 770 bp cDNA fragment was derived from the cytoplasmic portion of the EGF-R (ref. Libermann et al., 1985). Bg1II identifies 4 invariant bands of 7.0, 5.0, 3.5 and 1.2 kb and a two allele polymorphism with a band of either 10.6 kb (lane 1) or 9.4 kb (lane 3). An heterozygote individual is represented. The frequency was analyzed in 78 unrelated European Caucasians. Its chromosomal location was determined. Co-dominant segregation was demonstrated in three families of 12 individuals. A rare variant of 8.3 kb was seen in one chromosome out of the 144 examined. This allelic form has not yet been fully characterized.

  20. Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

    Energy Technology Data Exchange (ETDEWEB)

    Backer, Joseph M.

    2009-07-12

    In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need for information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for 1) rational patient stratification, 2) rapid monitoring of responses to therapy, and 3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg’s group at Stanford

  1. Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

    International Nuclear Information System (INIS)

    Backer, Joseph M.

    2009-01-01

    In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need for information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for (1) rational patient stratification, (2) rapid monitoring of responses to therapy, and (3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg's group at Stanford

  2. RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

    Science.gov (United States)

    ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.<...

  3. EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

    International Nuclear Information System (INIS)

    Jung, Kyung-Ho; Park, Jin Won; Paik, Jin-Young; Quach, Cung Hoa Thien; Choe, Yearn Seong; Lee, Kyung-Han

    2012-01-01

    Introduction: As direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging. Methods: Human epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to 99m Tc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice. Results: Whereas both 99m Tc-Av-EGF and 99m Tc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. 99m Tc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). 99m Tc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h. Conclusion: 99m Tc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.

  4. Hypoxia changes the expression of the epidermal growth factor (EGF) system in human hearts and cultured cardiomyocytes

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque Ahmed; Goetze, Jens Peter

    2012-01-01

    by treatment with trastuzumab (20 nM). This resulted in inhibition of cardiomyocyte proliferation, but interestingly only in hypoxic cells. Co-treatment of HL-1 cells with HB-EGF (10 nM) but not with NRG-1 (5 ng/ml) rescued the cardiomyocytes from HER2 inhibition. HL-1 cardiomyocytes exposed to hypoxia...... revealed nuclear translocation of activated MAPK and the activity of this downstream signaling molecule was decreased by HER2 inhibition (20 nM trastuzumab), and re-established by HB-EGF (10 nM). CONCLUSIONS/SIGNIFICANCE: Hypoxia in the human heart alters the expression of the EGF system. Mimicking the HER...

  5. Time-dependent changes of levels of endogenous epidermal growth factor in submandibular glands, in kidneys, and in urine in rats during systemic treatment with EGF

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Jørgensen, P E; Thulesen, J

    1998-01-01

    Exogenous EGF influences the levels of endogenous EGF differently in the submandibular glands (SMG) and the kidneys. The aim of the present study was to examine the time-dependent changes in levels of endogenous EGF during 1-4 weeks of EGF treatment.......Exogenous EGF influences the levels of endogenous EGF differently in the submandibular glands (SMG) and the kidneys. The aim of the present study was to examine the time-dependent changes in levels of endogenous EGF during 1-4 weeks of EGF treatment....

  6. Factors contributing to the immunogenicity of meningococcal conjugate vaccines

    Science.gov (United States)

    Bröker, Michael; Berti, Francesco; Costantino, Paolo

    2016-01-01

    ABSTRACT Various glycoprotein conjugate vaccines have been developed for the prevention of invasive meningococcal disease, having significant advantages over pure polysaccharide vaccines. One of the most important features of the conjugate vaccines is the induction of a T-cell dependent immune response, which enables both the induction of immune memory and a booster response after repeated immunization. The nature of the carrier protein to which the polysaccharides are chemically linked, is often regarded as the main component of the vaccine in determining its immunogenicity. However, other factors can have a significant impact on the vaccine's profile. In this review, we explore the physico-chemical properties of meningococcal conjugate vaccines, which can significantly contribute to the vaccine's immunogenicity. We demonstrate that the carrier is not the sole determining factor of the vaccine's profile, but, moreover, that the conjugate vaccine's immunogenicity is the result of multiple physico-chemical structures and characteristics. PMID:26934310

  7. Primary study on the clinical significance of measurement of epidermal growth factor (EGF) and NPY concentrations in human semen plasma

    International Nuclear Information System (INIS)

    Zhang Jiyun; Ning Yong

    2008-01-01

    Objective: To investigate the difference between the semen plasma contents of EGF and NPY in fertile and non-fertile males with the relevant sperm count and motility. Methods: Semen plasma contents of EGF and NPY were determined with RIA in 110 non-fertile males. Simultaneous semen analysis revealed (1) Group A, n=45, with normal sperm count, (2) Group B, n=34 low sperm count (0-20) x 10 6 /ml and (3) Group C n=31, with aspermia. White blood cell/HPF was examined in all the semen specimens and sperm motile rate and motility were examined in Group A specimens. Results: The semen plasma contents of EGF and NPY in non-fertile males were significantly higher than those in fertile males (P 1 x 10 6 /ml) were significantly lower than those in specimens with more white blood cells (P<0.05). Conclusion: Higher semen plasma contents of EGF and NPY might exert toxic effect on the sperms, contributing to the development of infertility. (authors)

  8. Reduced appearance of under-eye bags with twice-daily application of epidermal growth factor (EGF) serum: a pilot study.

    Science.gov (United States)

    Seidel, Rachel; Moy, Ronald L

    2015-04-01

    Under-eye bags are a common manifestation of age and a frequent complaint among patients who no longer feel youthful. Non-invasive topical agents are largely ineffective at reducing their appearance. We studied the ability of a topical serum containing epidermal growth factor (EGF) to minimize the appearance of under-eye bags. A single-center clinical trial was performed on eighteen volunteer male and female patients with under-eye bags. Subjects applied EGF serum to the infraorbital area twice daily for 12 weeks. At each visit, subjects were evaluated using clinical photography and written self-assessment. A grade on the Merz Infraorbital Hollowness Scale was also given and two independent, blind investigators assigned an Investigator's Global Assessment (IGA) score. At the trial's end, patients shared their final evaluation and perception of results with a questionnaire. Sixteen subjects completed the trial. The final average Merz grade was 1.63 (SEM = .273), statistically significantly lower than the mean baseline average of 2.06 (SEM = .232) (P = .0019). A reduction in average IGA score was also significant (Pbags as milder at the end of the trial compared to the first visit. Seven subjects reported greater satisfaction with their overall facial appearance. Of the subjects who had used other topical treatments in the past, two reported the serum to be "significantly better" and four said it was "better" in treating their under-eye bags. Our results offer evidence that topical EGF can reduce the appearance of under-eye bags.

  9. Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

    Science.gov (United States)

    Lee, Ming-Fen; Pan, Min-Hsiung; Chiou, Yi-Siou; Cheng, An-Chin; Huang, Han

    2011-11-09

    Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.

  10. Restoration of Circulating MFGE8 (Milk Fat Globule-EGF Factor 8) Attenuates Cardiac Hypertrophy Through Inhibition of Akt Pathway.

    Science.gov (United States)

    Deng, Ke-Qiong; Li, Jing; She, Zhi-Gang; Gong, Jun; Cheng, Wen-Lin; Gong, Fu-Han; Zhu, Xue-Yong; Zhang, Yan; Wang, Zhihua; Li, Hongliang

    2017-10-01

    Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting Mfge8 in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific Mfge8 overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either Mfge8 -knockout or Mfge8 -overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3β (glycogen synthase kinase-3β)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by Mfge8 deficiency but suppressed by Mfge8 overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of Mfge8 deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy. © 2017 American Heart Association, Inc.

  11. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    International Nuclear Information System (INIS)

    Shimura, Takaya; Higashiyama, Shigeki; Joh, Takashi; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi

    2012-01-01

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for

  12. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  13. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor.

    Science.gov (United States)

    Armant, D Randall; Kilburn, Brian A; Petkova, Anelia; Edwin, Samuel S; Duniec-Dmuchowski, Zophia M; Edwards, Holly J; Romero, Roberto; Leach, Richard E

    2006-02-01

    Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

  14. Gastric secretion, proinflammatory cytokines and epidermal growth factor (EGF) in the delayed healing of lingual and gastric ulcerations by testosterone.

    Science.gov (United States)

    Machowska, A; Brzozowski, T; Sliwowski, Z; Pawlik, M; Konturek, P C; Pajdo, R; Szlachcic, A; Drozdowicz, D; Schwarz, M; Stachura, J; Konturek, S J; Pawlik, W W

    2008-02-01

    Hormonal fluctuations are known to predispose ulceration of the upper gastrointestinal tract, but to date no comparative study of their effects on the healing of pre-existing ulcers in the oral cavity and stomach has been made. We studied the effects of depletion of testosterone and of EGF on the healing of acetic acid-induced ulcers using rats having undergone bilateral orchidectomy and/or salivectomy respectively. We measured alterations in gastric acid secretion and blood flow at ulcer margins, as well as plasma levels of testosterone, gastrin and the proinflammatory cytokines IL-1 beta and TNF-alpha. Testosterone (0.01-10 mg/kg/day i. m.) dose-dependently delayed oral and gastric ulcer healing. When applied in an optimal dose of 1 mg/kg/day, this hormone significantly raised gastric acid secretion and plasma IL-1 beta and TNF-alpha levels. Attenuation of plasma testosterone levels via bilateral orchidectomy inhibited gastric acid secretion and accelerated the healing of oral and gastric ulcers, while increasing plasma gastrin levels and these effects were reversed by testosterone. Salivectomy raised plasma testosterone levels, and delayed oral and gastric ulcer healing. Treatment of salivectomised animals with testosterone further inhibited ulcer healing, and this effect was counteracted by EGF. We propose that testosterone delays ulcer healing via a fall in blood flow at the ulcer margin, a rise in plasma levels of IL-1 beta and TNF-alpha and, in the case of gastric ulcers, an increase in gastric acid secretion. EGF released from the salivary glands plays an important role in limitation of the deleterious effects of testosterone on ulcer healing.

  15. Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

    Science.gov (United States)

    Bolnick, Alan D; Bolnick, Jay M; Kohan-Ghadr, Hamid-Reza; Kilburn, Brian A; Pasalodos, Omar J; Singhal, Pankaj K; Dai, Jing; Diamond, Michael P; Armant, D Randall; Drewlo, Sascha

    2017-06-01

    Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB

  16. Modulation of integrin-linked kinase (ILK expression in human oesophageal squamous cell carcinoma cell lines by the EGF and TGFβ1 growth factors

    Directory of Open Access Journals (Sweden)

    Veale Robin B

    2006-04-01

    Full Text Available Abstract Background Integrin-linked kinase (ILK is a ubiquitously expressed protein kinase that has emerged as one of the points of convergence between integrin- and growth factor-signalling pathways. Results In this study we identify the ILK isoform expressed in five human oesophageal squamous cell carcinoma cell lines of South African origin as ILK1, and demonstrate its cellular distribution. ILK expression, although similar in the majority of the cell lines, did show variation. Furthermore, the ILK expressed was shown to be catalytically functional. The effect of growth factors on ILK expression was examined. An increase in ILK expression, following EGF and TGFβ1 exposure, was a trend across all the five oesophageal carcinoma cell lines tested. Conclusion These results suggest that growth factor modulation of ILK expression relies on the internalisation/recycling of growth factor receptors and stimulation of the PI3K pathway, which may have implications with regards to cell adhesion and tumourigenesis.

  17. Serous carcinomatous component championed by heparin-binding EGF-like growth factor (HB-EGF) predisposing to metastasis and recurrence in stage I uterine malignant mixed mullerian tumor.

    Science.gov (United States)

    Zhang, Lei; Shimizu, David; Killeen, Jeffrey L; Honda, Stacey A; Lu, Di; Stanoyevitch, Alexander; Lin, Fritz; Wang, Beverly; Monuki, Edwin S; Carbone, Michele

    2016-07-01

    The stage I uterine malignant mixed mullerian tumor (MMMT) shows different potential for progression. We reason that MMMTs with high-grade carcinomatous component and positivity for HB-EGF are prone to recurrence/metastasis in the early stage. A retrospective clinical and histopathologic review with immunohistochemical staining for HB-EGF, EGFR, and integrin-α5 was performed for 62 surgically staged MMMT cases. Recurrence/metastasis (RM) is 6/18 (33%) in stage I disease. Of all the clinicopathologic variables and biomarkers analyzed for stage I MMMT, serous carcinomatous component (83% [5/6] versus 17% [1/12], P = .0015) and HB-EGF expression (100% [6/6] versus 50% [6/12], P=.0339) were significantly different between groups with RM and without RM. The presence of serous carcinoma in all stages was 83% (5/6) in stage I with RM, 8% (1/12) in stage I without RM, 20% (1/5) in stage II, 36.4% (8/22) in stage III and 64.7% (11/17) in stage IV; this was paralleled by HB-EGF expression of 100% (6/6), 50% (6/12), 40% (2/5), 50% (11/22) and 71% (12/17) with a correlation coefficient r=0.9131 (P=.027). HB-EGF and integrin-α5 were highly expressed in MMMTs bearing serous carcinoma component, compared to endometrioid and unclassifiable/miscellaneous subtypes (84.6%/47.6%/33.3%, P=.025 for HB-EGF; and 61.5%/42.9%/20.0%, P=.021 for integrin-α5). The EGFR positivity was comparable among the three subtypes (48.1%, 47.6% and 26.7%, P=.326). This study indicates that serous carcinomatous component championed by expression of HB-EGF predisposes to recurrence/metastasis in stage I MMMT. This process might involve integrin-α5 and does not seem to require overexpression of EGFR. Further study is required. Published by Elsevier Inc.

  18. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

    Science.gov (United States)

    Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2012-05-30

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation

  19. Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

    International Nuclear Information System (INIS)

    Schaudies, R.P.; Wray, H.L.

    1988-01-01

    We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells

  20. Improved expression of recombinant plant-made hEGF.

    Science.gov (United States)

    Thomas, David Rhys; Walmsley, Amanda Maree

    2014-11-01

    The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

  1. Factors Affecting the Pharmacology of Antibody–Drug Conjugates

    Directory of Open Access Journals (Sweden)

    Andrew T. Lucas

    2018-02-01

    Full Text Available Major advances in therapeutic proteins, including antibody–drug conjugates (ADCs, have created revolutionary drug delivery systems in cancer over the past decade. While these immunoconjugate agents provide several advantages compared to their small-molecule counterparts, their clinical use is still in its infancy. The considerations in their development and clinical use are complex, and consist of multiple components and variables that can affect the pharmacologic characteristics. It is critical to understand the mechanisms employed by ADCs in navigating biological barriers and how these factors affect their biodistribution, delivery to tumors, efficacy, and toxicity. Thus, future studies are warranted to better understand the complex pharmacology and interaction between ADC carriers and biological systems, such as the mononuclear phagocyte system (MPS and tumor microenvironment. This review provides an overview of factors that affect the pharmacologic profiles of ADC therapies that are currently in clinical use and development.

  2. Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

    Science.gov (United States)

    Heo, Jeongyeon; Kim, Jae Geun; Kim, Sunghwan; Kang, Hara

    2017-12-23

    Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy. © 2017 Wiley Periodicals, Inc.

  3. Cellular retention of radioactivity and increased radiation dose. Model experiments with EGF-dextran

    International Nuclear Information System (INIS)

    Sundberg, Aasa Liljegren; Blomquist, Erik; Carlsson, Joergen; Steffen, Ann-Charlott; Gedda, Lars

    2003-01-01

    Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with 125 I on different parts: [ 125 I]-EGF-dextran-[ 125 I], [ 125 I]-EGF-dextran and EGF-dextran-[ 125 I]. Comparisons were made with [ 125 I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that 131 I, instead of 125 I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/μg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose

  4. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Directory of Open Access Journals (Sweden)

    Shimura Takaya

    2012-05-01

    Full Text Available Abstract Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C, translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF and mutated HB-EGF (HB-EGF-mC, which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 % and in the cytoplasm only in 25 cases (26.0 %. The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P  Conclusions Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation might be crucial in gastric cancer invasion. HB-EGF-C nuclear translocation may offer a prognostic marker and a new molecular target for gastric cancer therapy.

  5. A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

    Directory of Open Access Journals (Sweden)

    Jacob Thomas

    Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

  6. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Science.gov (United States)

    2012-01-01

    Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Conclusions Both the function of HB-EGF as an EGFR ligand

  7. Stromal cell-derived factor-1α (SDF-1α/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    International Nuclear Information System (INIS)

    Porcile, Carola; Bajetto, Adriana; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio; Schettini, Gennaro

    2005-01-01

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1α treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1α induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer

  8. Age-related loss of EGF-receptor related protein (ERRP) in the aging colon is a potential risk factor for colon cancer.

    Science.gov (United States)

    Schmelz, Eva M; Levi, Edi; Du, Jianhua; Xu, Hu; Majumdar, Adhip P N

    2004-12-01

    Although in Fischer-344 rats, aging is associated with increased activation of EGF-receptor (EGFR) in mucosa of much of the gastrointestinal tract, including the colon, regulation of this process is poorly understood. We hypothesize that loss of suppressor of EGFR may partly be responsible for this process. To test this hypothesis, we examined the expression of EGFR related protein (ERRP), a recently identified negative regulator of EGFR, in the colonic mucosa during aging and following administration of the colonic carcinogen dimethylhydrazine (DMH) that resulted in the formation of aberrant crypt foci (ACF), which are considered to be precursor of adenoma and carcinoma. In Fischer-344 rats, aging is associated with increased activation of EGFR in the colonic mucosa, as evidenced by 30-35% increase in the levels of tyrosine phosphorylated EGFR in the proximal and distal colon of aged (20-22 months old) than in young (4-6 months old) rats. In contrast, the levels of ERRP in both regions of the colon of aged rats were decreased by 50-60%, compared to their younger counterparts. Administration of DMH, which induced a greater number of ACF in the colon of aged rats than in young animals, resulted in a corresponding reduction in ERRP in the colon. These results suggest that loss of ERRP expression is a common event during aging and early stages of chemically induced colon cancer. We also suggest that loss of ERRP could be a risk factor for developing colorectal cancer in the older population.

  9. Immunohistochemical and quantitative changes in salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer

    DEFF Research Database (Denmark)

    Christensen, M E; Hansen, H S; Poulsen, Steen Seier

    1996-01-01

    Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in t...... a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.......Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes....... The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated...

  10. Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway

    Directory of Open Access Journals (Sweden)

    Niluka Gunawardhana

    2018-01-01

    Full Text Available Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF, and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

  11. Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

    Science.gov (United States)

    Gunawardhana, Niluka; Jang, Sungil; Choi, Yun Hui; Hong, Youngmin A; Jeon, Yeong-Eui; Kim, Aeryun; Su, Hanfu; Kim, Ji-Hye; Yoo, Yun-Jung; Merrell, D Scott; Kim, Jinmoon; Cha, Jeong-Heon

    2017-01-01

    Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori -induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori -induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori -induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori -induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori -related hypergastrinemia and consequently gastric disease development, including gastric cancers.

  12. Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies

    NARCIS (Netherlands)

    Róg, T.; Murzyn, K.; Karttunen, M.E.J.; Pasenkiewicz-Gierula, M.

    2008-01-01

    A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine

  13. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    International Nuclear Information System (INIS)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-01-01

    Highlights: ► HBP sequence identified from HB-EGF has cell penetration activity. ► HBP inhibits the NF-κB dependent inflammatory responses. ► HBP directly blocks phosphorylation and degradation of IκBα. ► HBP inhibits nuclear translocation of NF-κB p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  14. A targetable HB-EGF-CITED4 axis controls oncogenesis in lung cancer.

    Science.gov (United States)

    Hsieh, C-H; Chou, Y-T; Kuo, M-H; Tsai, H-P; Chang, J-L; Wu, C-W

    2017-05-25

    Aberrant epidermal growth factor (EGF) receptor (EGFR) signaling contributes to neoplastic initiation and progression in lung. Mutated EGFR has become as an important therapeutic target in lung cancer, whereas targeted treatment is not available for wild-type EGFR or its ligands. In this study, we found that heparin-binding (HB)-EGF, a member of the EGF family, was highly expressed in a subset of lung cancer, proliferation of which was dependent on HB-EGF signaling. Silencing of HB-EGF with RNA interference inhibited cell cycle progression in lung cancer cells. We observed that, upon HB-EGF induction, CITED4 was induced through a signal transducer and activator of transcription 3 (STAT3)-dependent pathway, regulating cell proliferation. CITED4 interacted with MYC and potentiated MYC-mediated transactivation of the CCND1 promoter, leading to cell cycle progression. Correlation analysis revealed that HB-EGF and CITED4 were significantly positively associated in primary lung tumors, and expression of HB-EGF predicted a poor survival outcome in patients. In vitro and in vivo experiments revealed that pharmacological inhibition of HB-EGF with CRM197 significantly attenuated tumor cell growth. Thus, CITED4 functions as a molecular switch in HB-EGF-induced growth control, and HB-EGF provides a novel therapeutic target for lung cancer intervention.

  15. Effect of epidermal growth factor (EGF) on [3H]TdR incorporation into DNA in ad lib fed and fasted CD2F1 mice

    International Nuclear Information System (INIS)

    Scheving, L.A.; Tsai, T.H.; Scheving, L.E.; Hoke, W.S.

    1987-01-01

    The effect of EGF on the incorporation of [ 3 H]TdR into DNA (DNA synthesis) was determined in the esophagus, liver, pancreas, and kidney in mice standardized to 12 hours (hr) of light alternating with 12 hr of darkness. A question asked was whether intraperitoneally administered EGF could alter the circadian patterns of DNA synthesis in these organs. The most marked effects of EGF were: an increase in DNA synthesis but only after a specific duration of time after treatment, ranging from 8 to 23 hr, which differed for each tissue, a similarity in the response of the esophagus in both ad lib fed and fasted mice, but not in the response of the liver, where the stimulatory effect of EGF observed in fed mice was dramatically reduced in fasted ones, and an advance in the phasing of the circadian rhythm in DNA synthesis of the esophagus by about 12 hr. In addition, no sex differences in fasted animals were found under the conditions of this study

  16. Biodistribution of 99mTc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando

    1999-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG 1 ), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 μg/100 μCi of 99m Tc-labeled MAbs. Immunoreactivity of 99m Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 ± 2.08 %ID/g) and tumor (3.903 ± 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 ± 0.50 %ID/g and 2.19 ± 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the 99m Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued

  17. Biodistribution of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando E-mail: normando@ict.cim.sld.cu

    1999-04-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG{sub 1}), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled MAbs. Immunoreactivity of {sup 99m}Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 {+-} 2.08 %ID/g) and tumor (3.903 {+-} 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 {+-} 0.50 %ID/g and 2.19 {+-} 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the {sup 99m}Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.

  18. Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs

    Directory of Open Access Journals (Sweden)

    Yamashita Yasuhisa

    2009-12-01

    Full Text Available Abstract Objectives During ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17 are required for the activation of EGF receptor (EGFR, cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs. In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in Tace/Adam17 expression in cumulus cells of porcine COC during in vitro maturation. Methods Areg, Ereg, Tace/Adam17, Has2, Tnfaip6 and Ptgs2 mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope. Results When COCs were cultured with FSH and LH up to 2.5 h, Areg, Ereg and Tace/Adam17 mRNA were expressed in cumulus cells of COCs. Areg, Ereg and Tace/Adam17 gene expressions were not suppressed by PI3K inhibitor (LY294002, whereas PKA inhibitor (H89, p38 MAPK inhibitor (SB203580 and MEK inhibitor (U0126 significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of Has2, Tnfaip6 and Ptgs2 were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group. Conclusion The results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.

  19. Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

    Science.gov (United States)

    Suzuki, Keisuke; Mizushima, Hiroto; Abe, Hiroyuki; Iwamoto, Ryo; Nakamura, Haruki; Mekada, Eisuke

    2015-05-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  20. PET imaging of EGF receptors using [{sup 18}F]FBEM-EGF in a head and neck squamous cell carcinoma model

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weihua [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China); National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Niu, Gang; Lang, Lixin; Guo, Ning; Ma, Ying; Kiesewetter, Dale O.; Chen, Xiaoyuan [National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Backer, Joseph M. [SibTech Inc., Brookfield, CT (United States); Shen, Baozhong [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China)

    2012-02-15

    To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF). Cys-tagged EGF (cEGF) was labeled with {sup 18}F by coupling the free thiol group of the Cys tag with N-[2-(4-[{sup 18}F]fluorobenzamido)ethyl]maleimide ([{sup 18}F]FBEM) to form [{sup 18}F]FBEM-cEGF. Cell uptake, internalization and efflux of [{sup 18}F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results. The radiolabeling yield for [{sup 18}F]FBEM-cEGF was over 60%, based on starting [{sup 18}F]FBEM. [{sup 18}F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 {+-} 0.59 %ID/g at 30 min after injection. Significantly higher tumor uptake of [{sup 18}F]FBEM-cEGF (5.99 {+-} 1.61%ID/g at 30 min after injection, p < 0.01) and tumor/nontumor ratio were achieved by coinjection of 50 {mu}g of unlabeled EGF. Decreased liver uptake of [{sup 18}F]FBEM-cEGF was observed when unlabeled EGF was coadministered. With optimized liver blocking, [{sup 18}F]FBEM-cEGF has the potential to be used in a noninvasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity. (orig.)

  1. Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

    International Nuclear Information System (INIS)

    Lin, P.H.; Selinfreund, R.; Wharton, W.

    1986-01-01

    Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125 I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125 I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125 I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands

  2. The Role of Neurotrophic Factors Conjugated to Iron Oxide Nanoparticles in Peripheral Nerve Regeneration: In Vitro Studies

    Directory of Open Access Journals (Sweden)

    Ofra Ziv-Polat

    2014-01-01

    Full Text Available Local delivery of neurotrophic factors is a pillar of neural repair strategies in the peripheral nervous system. The main disadvantage of the free growth factors is their short half-life of few minutes. In order to prolong their activity, we have conjugated to iron oxide nanoparticles three neurotrophic factors: nerve growth factor (βNGF, glial cell-derived neurotrophic factor (GDNF, and basic fibroblast growth factor (FGF-2. Comparative stability studies of free versus conjugated factors revealed that the conjugated neurotrophic factors were significantly more stable in tissue cultures and in medium at 37°C. The biological effects of free versus conjugated neurotrophic factors were examined on organotypic dorsal root ganglion (DRG cultures performed in NVR-Gel, composed mainly of hyaluronic acid and laminin. Results revealed that the conjugated neurotrophic factors enhanced early nerve fiber sprouting compared to the corresponding free factors. The most meaningful result was that conjugated-GDNF, accelerated the onset and progression of myelin significantly earlier than the free GDNF and the other free and conjugated factors. This is probably due to the beneficial and long-acting effect that the stabilized conjugated-GDNF had on neurons and Schwann cells. These conclusive results make NVR-Gel enriched with conjugated-GDNF, a desirable scaffold for the reconstruction of severed peripheral nerve.

  3. On form factors of the conjugated field in the non-linear Schroedinger model

    Energy Technology Data Exchange (ETDEWEB)

    Kozlowski, K.K.

    2011-05-15

    Izergin-Korepin's lattice discretization of the non-linear Schroedinger model along with Oota's inverse problem provides one with determinant representations for the form factors of the lattice discretized conjugated field operator. We prove that these form factors converge, in the zero lattice spacing limit, to those of the conjugated field operator in the continuous model. We also compute the large-volume asymptotic behavior of such form factors in the continuous model. These are in particular characterized by Fredholm determinants of operators acting on closed contours. We provide a way of defining these Fredholm determinants in the case of generic paramaters. (orig.)

  4. HB-EGF expression as a potential biomarker of acquired middle ear cholesteatoma.

    Science.gov (United States)

    Xie, Shumin; Wang, Xiaoli; Ren, Hongmiao; Liu, Xiaoyu; Ren, Jihao; Liu, Wei

    2017-08-01

    The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma. To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation. A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed. On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

  5. Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

    Science.gov (United States)

    Zbinden, Aline; Browne, Shane; Altiok, Eda I; Svedlund, Felicia L; Jackson, Wesley M; Healy, Kevin E

    2018-05-01

    Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

  6. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    Energy Technology Data Exchange (ETDEWEB)

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-07-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

  7. SMAD4 loss enables EGF, TGF?1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

    OpenAIRE

    Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H.; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

    2016-01-01

    Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor ?1 (TGF?1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGF?1 and S100A8/A...

  8. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Szlachcic A

    2016-08-01

    Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

  9. Expression of polyhedrin-hEGF fusion protein in cultured cells and ...

    African Journals Online (AJOL)

    For mass production of human epidermal growth factor (hEGF), silkworm baculovirus expression vector system (BEVS) was adopted in this study. hEGF gene was in-frame fused with polyhedrin (Ph) gene under the control of Ph promoter and was used to co-transfect BmN cell with the modified. Bombyx mori baculovirus ...

  10. Risk factors for pneumococcal nasopharyngeal colonization before and after pneumococcal conjugate vaccination in persons with HIV

    DEFF Research Database (Denmark)

    Öbrink-Hansen, Kristina; Søgaard, Ole S; Harboe, Zitta B

    HIV-infected individuals have excess rates of invasive pneumococcal disease. We investigated risk factors for nasopharyngeal pneumococcal colonization at baseline and after 9 months in 96 HIV patients immunized twice with 7- valent pneumococcal conjugate vaccine ±1mg CPG 7909. In total, 22 patients...

  11. Requirement for Vibrio cholerae integration host factor in conjugative DNA transfer.

    Science.gov (United States)

    McLeod, Sarah M; Burrus, Vincent; Waldor, Matthew K

    2006-08-01

    The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to lambda integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

  12. DNA methylation dynamics in the rat EGF gene promoter after partial hepatectomy

    Directory of Open Access Journals (Sweden)

    Deming Li

    2014-06-01

    Full Text Available Epidermal growth factor (EGF, a multifunctional growth factor, is a regulator in a wide variety of physiological processes. EGF plays an important role in the regulation of liver regeneration. This study was aimed at investigating the methylation level of EGF gene throughout liver regeneration. DNA of liver tissue from control rats and partial hepatectomy (PH rats at 10 time points was extracted and a 354 bp fragment including 10 CpG sites from the transcription start was amplified after DNA was modified by sodium bisulfate. The result of sequencing suggested that methylation ratio of four CpG sites was found to be significantly changed when PH group was compared to control group, in particular two of them were extremely striking. mRNA expression of EGF was down-regulated in total during liver regeneration. We think that the rat EGF promoter region is regulated by variation in DNA methylation during liver regeneration.

  13. * Hierarchically Structured Electrospun Scaffolds with Chemically Conjugated Growth Factor for Ligament Tissue Engineering.

    Science.gov (United States)

    Pauly, Hannah M; Sathy, Binulal N; Olvera, Dinorath; McCarthy, Helen O; Kelly, Daniel J; Popat, Ketul C; Dunne, Nicholas J; Haut Donahue, Tammy Lynn

    2017-08-01

    The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue-engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macroscale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF)-conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF were assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles), or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles), and ligament-specific tissue formation was assessed in comparison to non-CTGF-conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament-specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

  14. Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization.

    Science.gov (United States)

    Inoue, Yuki; Shimazawa, Masamitsu; Nakamura, Shinsuke; Takata, Shinsuke; Hashimoto, Yuhei; Izawa, Hiroshi; Masuda, Tomomi; Tsuruma, Kazuhiro; Sakaue, Tomohisa; Nakayama, Hironao; Higashiyama, Shigeki; Hara, Hideaki

    2018-01-01

    The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis. We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration. © 2017 American Heart Association, Inc.

  15. Changes in proHB-EGF expression after functional activation of the immune system cells

    Directory of Open Access Journals (Sweden)

    T. O. Chudina

    2017-12-01

    Full Text Available The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197 and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.

  16. Arabidopsis TCP Transcription Factors Interact with the SUMO Conjugating Machinery in Nuclear Foci

    Directory of Open Access Journals (Sweden)

    Magdalena J. Mazur

    2017-11-01

    Full Text Available In Arabidopsis more than 400 proteins have been identified as SUMO targets, both in vivo and in vitro. Among others, transcription factors (TFs are common targets for SUMO conjugation. Here we aimed to exhaustively screen for TFs that interact with the SUMO machinery using an arrayed yeast two-hybrid library containing more than 1,100 TFs. We identified 76 interactors that foremost interact with the SUMO conjugation enzyme SCE1 and/or the SUMO E3 ligase SIZ1. These interactors belong to various TF families, which control a wide range of processes in plant development and stress signaling. Amongst these interactors, the TCP family was overrepresented with several TCPs interacting with different proteins of the SUMO conjugation cycle. For a subset of these TCPs we confirmed that the catalytic site of SCE1 is essential for this interaction. In agreement, TCP1, TCP3, TCP8, TCP14, and TCP15 were readily SUMO modified in an E. coli sumoylation assay. Strikingly, these TCP-SCE1 interactions were found to redistribute these TCPs into nuclear foci/speckles, suggesting that these TCP foci represent sites for SUMO (conjugation activity.

  17. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  18. EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

    Science.gov (United States)

    Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Identification of a novel EGF-sensitive cell cycle checkpoint

    International Nuclear Information System (INIS)

    Walker, Francesca; Zhang Huihua; Burgess, Antony W.

    2007-01-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells

  20. Conjugated linoleic acid as a potential protective factor in prevention of breast cancer 

    Directory of Open Access Journals (Sweden)

    Agnieszka Białek

    2013-01-01

    Full Text Available Cancers are the second leading cause of deaths in Poland, among both women and men. Breast cancer is the malignancy most frequently diagnosed in women. In 2008 mammary cancer was diagnosed in up to 14 500 patients. It is also the second most common cause of cancer deaths among women in our country. Although the etiology of most cases of this disease is not known, risk factors include a variety of nutritional factors. The amount of fat consumed in the diet and the quantity and quality of fatty acids are especially crucial. Among fatty acids to which great importance in modification of cancer risk is attributed are conjugated linoleic acid. Conjugated linoleic acids (CLA are a group of positional and geometric isomers of linoleic acid, with a conjugated double bond system in the carbon chain. The main natural source of them is milk and dairy products and meat of different species of ruminants, in which cis-9, trans-11 octadecadienoic acid (rumenic acid occurs in the largest quantities, constituting over 90�0of the total pool of CLA. Another important isomer is trans-10, cis-12 octadecadienoic acid, which occurs with rumenic acid in dietary supplements, usually in the ratio 1:1. Surveys conducted show their possible health promoting effects in obesity, atherosclerosis, cardiovascular diseases, osteoporosis, diabetes, insulin resistance, inflammation, and various types of cancer, especially breast cancer. 

  1. A Gly1127Ser mutation in an EGF-like domain of the Fibrillin-I gene is a risk factor for ascending aortic aneurysm and dissection

    Energy Technology Data Exchange (ETDEWEB)

    Francke, U.; Berg, M.A.; Tynan, K. [Stanford Univ. Medical Center, CA (United States)] [and others

    1995-06-01

    Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromsomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EFG-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly 1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life. 33 refs., 6 figs.

  2. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines.

    DEFF Research Database (Denmark)

    Tørring, Niels; Sørensen, Boe Sandahl; Nexø, Ebba

    2000-01-01

    BACKGROUND: The proliferation of androgen-independent prostate cancer cell lines has previously been shown to be influenced by an autocrine loop of the epidermal growth factor (EGF) system. This observation has alerted us to study the expression of ligands and receptors from the EGF......-system in prostate cell lines. METHODS: The expression of the EGF system was determined by quantitative RT-PCR and ELISA in the normal prostate epithelial cell line (PNT1A), in the androgen sensitive-(LNCaP), and the androgen-independent (DU145 and PC3) prostate cancer cell lines. RESULTS: The expression of m...... which exhibit low expression of HER1. Similar results were obtained by ELISA. CONCLUSIONS: The data indicates a selective up-regulation of a subclass of ligands of the EGF-system in androgen-independent prostate cancer cell lines. We suggest this could be a mechanism to escape androgen dependence...

  3. Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

    OpenAIRE

    McLeod, Sarah M.; Burrus, Vincent; Waldor, Matthew K.

    2006-01-01

    The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. I...

  4. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

    DEFF Research Database (Denmark)

    Erba, Elisabetta Boeri; Matthiesen, Rune; Bunkenborg, Jakob

    2007-01-01

    Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium p...

  5. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ebi, Masahide [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan)

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  6. TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    International Nuclear Information System (INIS)

    Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2010-01-01

    Research highlights: → TGFβ induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. → TGFβ induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. → TGFβ enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. → Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGFβ. → ADAM17 may play a crucial role in this TGFβ-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF

  7. Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

    Directory of Open Access Journals (Sweden)

    Timothy L Haskett

    2018-03-01

    Full Text Available Tripartite integrative and conjugative elements (ICE3 are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

  8. Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

    Science.gov (United States)

    Haskett, Timothy L; Terpolilli, Jason J; Ramachandran, Vinoy K; Verdonk, Callum J; Poole, Phillip S; O'Hara, Graham W; Ramsay, Joshua P

    2018-03-01

    Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

  9. 9-Hydroxystearic acid interferes with EGF signalling in a human colon adenocarcinoma

    International Nuclear Information System (INIS)

    Calonghi, Natalia; Pagnotta, Eleonora; Parolin, Carola; Tognoli, Cristina; Boga, Carla; Masotti, Lanfranco

    2006-01-01

    The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal

  10. Targeted delivery of polyamidoamine-paclitaxel conjugate functionalized with anti-human epidermal growth factor receptor 2 trastuzumab

    Directory of Open Access Journals (Sweden)

    Ma P

    2015-03-01

    Full Text Available Pengkai Ma,1 Xuemei Zhang,1 Ling Ni,2 Jinming Li,2 Fengpu Zhang,1 Zheng Wang,1 Shengnan Lian,1 Kaoxiang Sun1 1School of Pharmacy, Yantai University, Yantai, Shandong Province, People’s Republic of China; 2State Key Laboratory of Long-acting and Targeting Drug Delivery System, Yantai, Shandong Province, People’s Republic of China Background: Antibody-dendrimer conjugates have the potential to improve the targeting and release of chemotherapeutic drugs at the tumor site while reducing adverse side effects caused by drug accumulation in healthy tissues. In this study, trastuzumab (TMAB, which binds to human epidermal growth factor receptor 2 (HER2, was used as a targeting agent in a TMAB-polyamidoamine (PAMAM conjugate carrying paclitaxel (PTX specifically to cells overexpressing HER2. Methods: TMAB was covalently linked to a PAMAM dendrimer via bifunctional polyethylene glycol (PEG. PTX was conjugated to PAMAM using succinic anhydride as a cross-linker, yielding TMAB-PEG-PAMAM-PTX. Dynamic light scattering and transmission electron microscopy were used to characterize the conjugates. The cellular uptake and in vivo biodistribution were studied by fluorescence microscopy, flow cytometry, and Carestream In Vivo FX, respectively. Results: Nuclear magnetic resonance spectroscopy demonstrated that PEG, PTX, fluorescein isothiocyanate, and cyanine7 were conjugated to PAMAM. Ultraviolet-visible spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that TMAB was conjugated to PEG-PAMAM. Dynamic light scattering and transmission electron microscopy measurements revealed that the different conjugates ranged in size between 10 and 35 nm and had a spherical shape. In vitro cellular uptake demonstrated that the TMAB-conjugated PAMAM was taken up by HER2-overexpressing BT474 cells more efficiently than MCF-7 cells that expressed lower levels of HER2. Co-localization experiments indicated that TMAB-conjugated PAMAM was

  11. Decreased autophosphorylation of EGF receptor in insulin-deficient diabetic rats

    International Nuclear Information System (INIS)

    Okamoto, M.; Kahn, C.R.; Maron, R.; White, M.F.

    1988-01-01

    The authors have previously reported that despite an increase in receptor concentration, there is a decrease in autophosphorylation and tyrosine kinase activity of the insulin receptor in insulin-deficient diabetic rats. To determine if other tyrosine kinases might be altered, they have studied the epidermal growth factor (EGF) receptor kinase in wheat germ agglutinin-purified, Triton X-100-solubilized liver membranes from streptozotocin (STZ)-induced diabetic rats and the insulin-deficient BB rat. They find that autophosphorylation of EGF receptor is decreased in proportion to the severity of the diabetic state in STZ rats with a maximal decrease of 67%. A similar decrease in autophosphorylation was observed in diabetic BB rats that was partially normalized by insulin treatment. Separation of tryptic phosphopeptides by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. A parallel decrease in EGF receptor phosphorylation was also found by immunoblotting with an antiphosphotyrosine antibody. EGF receptor concentration, determined by Scatchard analysis of 125 I-labeled EGF binding, was decreased by 39% in the STZ rat and 27% in the diabetic BB rat. Thus autophosphorylation of EGF receptor, like that of the insulin receptor, is decreased in insulin-deficient rat liver. In the case of EGF receptor, this is due in part to a decrease in receptor number and in part to a decrease in the specific activity of the kinase

  12. Pretreatment with oleic acid accelerates the entrance into the mitotic cycle of EGF-stimulated fibroblasts.

    Science.gov (United States)

    Zugaza, J L; Casabiell, X A; Bokser, L; Eiras, A; Beiras, A; Casanueva, F F

    1995-07-01

    We have previously demonstrated that pretreatment of several cell lines with cis-unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acids accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.

  13. SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

    Science.gov (United States)

    Thomas, J Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R; Moos, Malcolm

    2016-01-01

    In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

  14. Chronic cerebral hypoperfusion-induced impairment of Aβ clearance requires HB-EGF-dependent sequential activation of HIF1α and MMP9.

    Science.gov (United States)

    Ashok, Anushruti; Rai, Nagendra Kumar; Raza, Waseem; Pandey, Rukmani; Bandyopadhyay, Sanghamitra

    2016-11-01

    Chronic cerebral hypoperfusion (CCH) manifests Alzheimer's Disease (AD) neuropathology, marked by increased amyloid beta (Aβ). Besides, hypoxia stimulates Heparin-binding EGF-like growth factor (HB-EGF) mRNA expression in the hippocampus. However, involvement of HB-EGF in CCH-induced Aβ pathology remains unidentified. Here, using Bilateral Common Carotid Artery Occlusion mouse model, we explored the mechanism of HB-EGF regulated Aβ induction in CCH. We found that HB-EGF inhibition suppressed, while exogenous-HB-EGF triggered hippocampal Aβ, proving HB-EGF-dependent Aβ increase. We also detected that HB-EGF affected the expression of primary Aβ transporters, receptor for advanced glycation end-products (RAGE) and lipoprotein receptor-related protein-1 (LRP-1), indicating impaired Aβ clearance across the blood-brain barrier (BBB). An HB-EGF-dependent loss in BBB integrity supported impaired Aβ clearance. The effect of HB-EGF on Amyloid Precursor Protein pathway was relatively insignificant, suggesting a lesser effect on Aβ generation. Delving into BBB disruption mechanism demonstrated HB-EGF-mediated stimulation of Matrix metalloprotease-9 (MMP9), which affected BBB via HB-EGF-ectodomain shedding and epidermal growth factor receptor activation. Examining the intersection of HB-EGF-regulated pathway and hypoxia revealed HB-EGF-dependent increase in transcription factor, Hypoxia-inducible factor-1alpha (HIF1α). Further, via binding to hypoxia-responsive elements in MMP9 gene, HIF1α stimulated MMP9 expression, and therefore appeared as a prominent intermediary in HB-EGF-induced BBB damage. Overall, our study reveals the essential role of HB-EGF in triggering CCH-mediated Aβ accumulation. The proposed mechanism involves an HB-EGF-dependent HIF1α increase, generating MMP9 that stimulates soluble-HB-EGF/EGFR-induced BBB disintegration. Consequently, CCH-mediated hippocampal RAGE and LRP-1 deregulation together with BBB damage impair Aβ transport and clearance

  15. EGF-induced stimualtion of EGF-receptor synthesis in human cytotrophoblasts and A431 cells

    International Nuclear Information System (INIS)

    DePalo, L.; Basu, A.; Das, M.

    1987-01-01

    EGF-receptor is a transmembrane glycoprotein whose intracellular degradation is known to be enhanced by EGF. The authors tested whether the receptor is replenished during this process by an enhanced rate of synthesis. Human A431 epidermoid carcinoma cells, and primary cultures of human placental cytotrophoblasts were used in these studies. Cells were labeled with 35 S-methionine, and EGF-receptor biosynthesis was quantitated by immunoprecipitation using a monoclonal anti-EGF-receptor antibody. EGF stimulated receptor biosynthesis at concentrations of 0.1-1 nM. The effect was seen within 2 h of EGF addition. The maximal stimulatory effect was modest in A431 (∼ 2-fold), but marked in the cytotrophoblasts (>5-fold). At EGF concentrations higher than 3 nM, the stimulatory effect was abolished. In contrast, the effect of EGF on receptor degradation is negligible at low subnanomolar concentrations, and is pronounced only at saturating concentrations. These results show that occupation of the cell surface EGF-receptor by its ligand can lead to production of more receptor protein, thus counterbalancing the negative effect on receptor degradation. At low subnanomolar (mitogenic) concentrations of EGF the stimulator effect on receptor synthesis is likely to predominate over the effect on receptor degradation

  16. Direct astatination of a tumour-binding protein, human epidermal growth factor, using nido-carborane as a prosthetic group

    International Nuclear Information System (INIS)

    Sjoestroem, A.; Carlsson, J.; Lundqvist, H.; Koziorowski, J.

    2003-01-01

    A method for direct astatine labeling of proteins has been investigated. Binding sites for astatine were created by coupling of a nido-carborane derivative to a protein, the human epidermal growth factor (hEGF), using two different conjugation methods - by glutaraldehyde cross-linking or by introduction of sulfohydryl groups by Traut's reagent with subsequent linking of ANC-1 with m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester. The conjugates were astatinated using the Chloramine-T method in high yield. The best labeling was obtained by the glutaraldehyde conjugate with an average yield of 68 ± 9%. In vitro stability tests indicated that the glutaraldehyde conjugated label was as stable as hEGF labeled with astatobenzoate. (author)

  17. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

    International Nuclear Information System (INIS)

    Semino, Carlos E.; Kamm, Roger D.; Lauffenburger, Douglas A.

    2006-01-01

    We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures

  18. Optical Molecular Imaging of Epidermal Growth Factor Receptor Expression to Improve Detection of Oral Neoplasia

    Directory of Open Access Journals (Sweden)

    Nitin Nitin

    2009-06-01

    Full Text Available Background: The development of noninvasive molecular imaging approaches has the potential to improve management of cancer. Methods: In this study, we demonstrate the potential of noninvasive topical delivery of an epidermal growth factor-Alexa 647 (EGF-Alexa 647 conjugate to image changes in epidermal growth factor receptor expression associated with oral neoplasia. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of EGF-Alexa 647 in a variety of model systems, including cells, three-dimensional tissue cultures, and intact human tissue specimens using wide-field and high-resolution fluorescence imaging. Data were collected from 17 different oral cancer patients: eight pairs of normal and abnormal biopsies and nine resected tumors were examined. Results: The EGF-dye conjugate can be uniformly delivered throughout the oral epithelium with a penetration depth exceeding 500 µm and incubation time of less than 30 minutes. After EGF-Alexa 647 incubation, the presence of oral neoplasia is associated with a 1.5- to 6.9-fold increase in fluorescence contrast compared with grossly normal mucosa from the same patient with both wide-field and high-resolution fluorescence imaging. Conclusions: Results illustrate the potential of EGF-targeted fluorescent agents for in vivo molecular imaging, a technique that may aid in the diagnosis and characterization of oral neoplasia and allow real-time detection of tumor margins.

  19. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration

    Science.gov (United States)

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. PMID:26464832

  20. Bioresponsive release of insulin-like growth factor-I from its PEGylated conjugate.

    Science.gov (United States)

    Braun, Alexandra C; Gutmann, Marcus; Mueller, Thomas D; Lühmann, Tessa; Meinel, Lorenz

    2018-06-10

    PEGylation of protein ligands, the attachment of polyethylene glycol (PEG) polymers to a therapeutic protein, increases therapeutics' half-life but frequently comes at the cost of reduced bioactivity. We are now presenting a bioinspired strategy leading out of this dilemma. To this end, we selected a position within insulin-like growth factor I (IGF-I) for decoration with a PEG 30kDa -modified protease-sensitive peptide linker (PSL) using a combination of enzymatic and chemical bioorthogonal coupling strategies. The PSL sequence responded to matrix metalloproteinases (MMP) to provide a targeted release in diseased tissue. The IGF-PSL-PEG conjugate had different binding protein affinity, cell proliferation, and endocytosis patterns as compared to the wild type. Exposure of the conjugate to elevated levels of activated MMPs, as present in inflamed tissues, fully reestablished the wild type properties through effective PSL cleavage. In conclusion, this bioinspired approach provided a blueprint for PEGylated therapeutics combining the pharmacokinetic advantages of PEGylation, while locally restoring the full suite of biological potential of therapeutics. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

    Science.gov (United States)

    Tian, Baomin; Wong, Wah Yau; Uger, Marni D; Wisniewski, Pawel; Chao, Heman

    2017-01-01

    Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MS E peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[( N -maleimidopropionamido)-diethyleneglycol] ester (SM(PEG) 2 ), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol

  2. Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2

    Directory of Open Access Journals (Sweden)

    Baomin Tian

    2017-08-01

    Full Text Available Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs. In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21 and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody–urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3 pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido-diethyleneglycol] ester (SM(PEG2, which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis

  3. A functional EGF+61 polymorphism is associated with severity of obstructive sleep apnea.

    Science.gov (United States)

    Ding, Qunli; Cao, Chao; Chen, Zhongbo; Tabusi, Mahebali; Chen, Li; Deng, Zaichun

    2015-05-01

    Involvement of epidermal growth factor (EGF) is reported in diseases caused by hypoxia. Its functional polymorphism may alter its transcription, affecting EGF expression, contributing to obstructive sleep apnea (OSA). The aim of this study was to investigate associations of EGF+61 polymorphism and risk of OSA. Two hundred two participants were enrolled in this case-control study. DNA was extracted from peripheral blood, and EGF 61A/G polymorphism was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. No significant association between EGF 61 A/G polymorphism and risk of OSA was observed in any of the gene models tested (AA vs. GG: OR = 0.97, 95% CI = 0.37-2.55; P = 0.95). However, compared with GG genotype, AG genotype associated with decreased risk of severe OSA (AG vs. GG: OR = 0.32, 95% CI = 0.11-0.94). Our study showed that AG genotype has a protective effect on OSA patients against severe disease, although EGF 61A/G polymorphisms have no role on the risk of the disease. Additional large studies should further validate our findings.

  4. HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration

    Science.gov (United States)

    Wan, Jin; Ramachandran, Rajesh; Goldman, Daniel

    2011-01-01

    Summary Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we report that heparin-binding epidermal-like growth factor (HB-EGF) is rapidly induced in MG residing at the injury site and that proHB-EGF ectodomain shedding is necessary for retina regeneration. Remarkably, HB-EGF stimulates the formation of multipotent MG-derived progenitors in the uninjured retina. We show that HB-EGF mediates its effects via an EGFR/MAPK signal transduction cascade that regulates the expression of regeneration-associated genes, like ascl1a and pax6b. We also uncover an HB-EGF/Ascl1a/Notch/hb-egfa signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/β-catenin signaling cascade that controls progenitor proliferation. These data provide a link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggest strategies for stimulating retina regeneration in mammals. PMID:22340497

  5. EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis.

    Science.gov (United States)

    Feng, Y; Dai, X; Li, X; Wang, H; Liu, J; Zhang, J; Du, Y; Xia, L

    2012-10-01

    Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self-renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b-FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self-renewal. Colon CSCs were cultured in serum-free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence-activated cell sorting and western blotting. Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi-1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal-regulated kinase 1/2 (ERK 1/2). This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer. © 2012 Blackwell Publishing Ltd.

  6. Odin (ANKS1A modulates EGF receptor recycling and stability.

    Directory of Open Access Journals (Sweden)

    Jiefei Tong

    Full Text Available The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.

  7. Effects of structurally stabilized EGF and bFGF on wound healing in type I and type II diabetic mice.

    Science.gov (United States)

    Choi, Seong Mi; Lee, Kyoung-Mi; Kim, Hyun Jung; Park, Ik Kyu; Kang, Hwi Ju; Shin, Hang-Cheol; Baek, Dawoon; Choi, Yoorim; Park, Kwang Hwan; Lee, Jin Woo

    2018-01-15

    Diabetes mellitus comprises a multiple metabolic disorder that affects millions of people worldwide and consequentially poses challenges for clinical treatment. Among the various complications, diabetic ulcer constitutes the most prevalent associated disorder and leads to delayed wound healing. To enhance wound healing capacity, we developed structurally stabilized epidermal growth factor (ST-EGF) and basic fibroblast growth factor (ST-bFGF) to overcome limitations of commercially available EGF (CA-EGF) and bFGF (CA-bFGF), such as short half-life and loss of activity after loading onto a matrix. Neither ST-EGF nor ST-bFGF was toxic, and both were more stable at higher temperatures than CA-EGF and CA-bFGF. We loaded ST-EGF and ST-bFGF onto a hyaluronate-collagen dressing (HCD) matrix, a biocompatible carrier, and tested the effectiveness of this system in promoting wound healing in a mouse model of diabetes. Wounds treated with HCD matrix loaded with 0.3 μg/cm 2 ST-EGF or 1 μg/cm 2 ST-bFGF showed a more rapid rate of tissue repair as compared to the control in type I and II diabetes models. Our results indicate that an HDC matrix loaded with 0.3 μg/cm 2 ST-EGF or 1 μg/cm 2 ST-bFGF can promote wound healing in diabetic ulcers and are suitable for use in wound dressings owing to their stability for long periods at room temperature. Various types of dressing materials loaded with growth factors, such as VEGF, EGF, and bFGF, are widely used to effect wound repair. However, such growth factor-loaded materials have several limitations for use as therapeutic agents in healing-impaired diabetic wounds. To overcome these limitations, we have developed new materials containing structurally stabilized EGF (ST-EGF) and bFGF (ST-bFGF). To confirm the wound healing capacity of newly developed materials (ST-EGF and ST-bFGF-loaded hyaluronate-collagen dressing [HCD] matrix), we applied these matrices in type I and type II diabetic wounds. Notably, these matrices were

  8. Nitric oxide generated by ionizing radiation and EGF is implicated in EGF receptor phosphorylation in A549 lung carcinoma cells

    International Nuclear Information System (INIS)

    Park, In Chul; Lee, Hyung Chahn; Rhee, Chang Hun; Hong, Seok Il

    2004-01-01

    Although it has been demonstrated that ionizing radiation (IR) control various cell functions in a different cell types, the mechanisms of its action via NO are not well understood. NO may potentially affect every type of mammalian cells, owing to its ubiquitous production and participate in the control of cell proliferation in a great variety of cell types. The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of Mr 170,000. When EGF binds to its receptor, the receptor is dimerized and autophosphorylated at the carboxyl-terminal tyrosine 992, 1608, 1086, 1148 and 1173. This phosphorylated receptor initiates a series of signal tranduction events through interacting proteins of SH2 family including Shc, Grb2 and Sos, which in turn trigger ativation of MAPK cascades. Although the number of signaling events mediated by IR-induced NO is growing, it is still unclear how NO activate cellular signaling events. Thus, we examined the effect of NO on cellular phosphorylation and found that NO was produced by ionizing radiation in A549 lung adenocarcinoma cells and enhances the unique tyrosine phosphorylation on EGF receptor

  9. Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus

    Science.gov (United States)

    Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.

    2016-01-01

    Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus. PMID:27243144

  10. Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Zandi, Nahid Karimi; Rasmussen, Mikkel Aabech

    2017-01-01

    It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the opti......It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action...... of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus-oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0-100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined...

  11. Synthesis and Characterization of Nanodiamond-Growth Factor Complexes Towards Applications in Oral Implantation and Regenerative Medicine.

    Science.gov (United States)

    Bang, Julie; Ting, Caleb; Wang, Peter; Kim, Ted; Wang, Kenneth; Kee, Theodore; Miya, Darron; Ho, Dean; Lee, Dong-Keun

    2018-02-19

    Current challenges in the field of regenerative medicine include the need to deliver sustained concentrations of growth factors and genes that are required to induce the repair of deficient tissues. Enhancement of drug delivery and uptake may result in improved growth factor efficacy. Nanodiamonds (NDs) were explored as potential growth factor delivery agents due to the many favorable properties that they possess. For example, ND's biocompatibility has been extensively validated pre-clinically. In addition, they can be scalably produced through impact events such as detonation. They possess notably faceted surfaces with diverse electrostatic properties that allow the rapid formation of growth factor complexes. In this study, a complex based on NDs conjugated to epidermal growth factor (EGF) functionalized with Alexa Fluor 488 (ND-EGF) was developed. ND-EGF was comprehensively evaluated using dynamic light scattering and zeta potential analysis. Furthermore, the NDs were capable of eluting EGF in a sustained fashion. Therefore, ND-EGF may serve as a promising nano-biomaterial for sustained growth factor elution.

  12. Changes of serum and chorion-villi contents of EGF in early pregnant women undergone artificial abortion

    International Nuclear Information System (INIS)

    Li Suping; Wu Xiaohua; Li Hui

    2008-01-01

    Objective: To investigate the changes of serum and chorion-villi contents of EGF in pregnant women undergone artificial abortion with drug (mifepristone) or surgery (curettage). Methods: Serum epidermal growth factor (EGF), E 2 , progesterone levels changes as well as chorion-villi EGF contents were measured with RIA in 36 pregnant women with drug abortion (before and after mifepristone 25mg bid x 3 days), 30 pregnant women undergone curettage (determined twice, 3 days apart) and 32 controls (serum only). Results: Serum EGF, E 2 , and progesterone contents in all pregnant women were significantly higher than those in controls (P<0.01). The chorion-villi contents of EGF in patients undergone drug abortion were significantly lower than those in patients undergone curettage (P<0.05). Both serum EGF and progesterone contents dropped after 3 days treatment with mifepristone (vs those in curettage group, P<0.05). Conclusion: Mifepristone might exert the effect of abortion through decrease of EGF levels, which was detrimental to fetus growth. (authors)

  13. Rational design of an EGF-IL18 fusion protein: Implication for developing tumor therapeutics

    International Nuclear Information System (INIS)

    Lu Jianxin; Peng Ying; Meng Zhefeng; Jin Liqin; Lu Yongsui; Guan Minxin

    2005-01-01

    Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly 4 Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-γ to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics

  14. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  15. Role of apoptosis-inducing factor (AIF in programmed nuclear death during conjugation in Tetrahymena thermophila

    Directory of Open Access Journals (Sweden)

    Endoh Hiroshi

    2010-02-01

    Full Text Available Abstract Background Programmed nuclear death (PND, which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation. In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation. Results We focused on the role of mitochondrial apoptosis-inducing factor (AIF during PND in Tetrahymena. The disruption of AIF delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA fragmentation. AIF is localized in Tetrahymena mitochondria and is released into the macronucleus prior to nuclear condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering. Conclusions Our results suggest that Tetrahymena AIF plays an important role in the degradation of DNA at an early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation pathway is widely conserved among eukaryotes.

  16. Using Chebyshev polynomials and approximate inverse triangular factorizations for preconditioning the conjugate gradient method

    Science.gov (United States)

    Kaporin, I. E.

    2012-02-01

    In order to precondition a sparse symmetric positive definite matrix, its approximate inverse is examined, which is represented as the product of two sparse mutually adjoint triangular matrices. In this way, the solution of the corresponding system of linear algebraic equations (SLAE) by applying the preconditioned conjugate gradient method (CGM) is reduced to performing only elementary vector operations and calculating sparse matrix-vector products. A method for constructing the above preconditioner is described and analyzed. The triangular factor has a fixed sparsity pattern and is optimal in the sense that the preconditioned matrix has a minimum K-condition number. The use of polynomial preconditioning based on Chebyshev polynomials makes it possible to considerably reduce the amount of scalar product operations (at the cost of an insignificant increase in the total number of arithmetic operations). The possibility of an efficient massively parallel implementation of the resulting method for solving SLAEs is discussed. For a sequential version of this method, the results obtained by solving 56 test problems from the Florida sparse matrix collection (which are large-scale and ill-conditioned) are presented. These results show that the method is highly reliable and has low computational costs.

  17. Reduction of Burn Progression with Topical Delivery of (Antitumor Necrosis Factor-alpha )-Hyaluronic Acid Conjugates

    Science.gov (United States)

    2012-01-01

    antibody conjugation to HA The conjugation chemistry followed a method previously developed in our laboratory. Briefly, HA (12 mg) was modi - fied...Webster MW, McGill JB, Schwartz SL. Promotion and acceleration of diabetic ulcer healing by arginine-glycine-aspartic acid (RGD) peptide matrix. RGD...Study Group. Diabetes Care 1995; 18: 39–46. 32. Ho-Asjoe M, Chronnell CM, Frame JD, Leigh IM, Carver N. Immunohistochemical analysis of burn depth. J

  18. Diagnostic of tumours of epithelial origin with the monoclonal antibody IOR EGF/R3 murino

    International Nuclear Information System (INIS)

    Ramos, M.

    1997-01-01

    Despite of the advantages on anti tumoral therapy, the cancer of epithelial origin constitutes one of the first causes of death worldwide. That kind of tumors have a 10-30-fold overexpression of the epidermal growth factor receptor (EGFr). Monoclonal antibody ior egf/r3 is a lgG2a, which recognizes the epidermal growth factor receptor. The aim of the present work was the evaluate the diagnostic efficacy of the 99m Tc-labelled ior egf/r3 for the detection of epithelial derived tumors, its metastasis and its recurrences

  19. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

    DEFF Research Database (Denmark)

    Chen, Yunhao; Low, Teck-Yew; Choong, Lee-Yee

    2007-01-01

    With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified...

  20. Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii × N. sanderae and transfer to honey by bees

    NARCIS (Netherlands)

    Helsper, J.P.F.G.; Ruyter-Spira, C.P.; Kwakman, P.H.S.; Bleeker, W.K.; Keizer, L.C.P.; Bade, J.B.; Velde, te A.A.; Zaat, S.A.J.; Verbeek, M.; Creemers-Molenaar, J.

    2011-01-01

    Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be

  1. Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees

    NARCIS (Netherlands)

    Helsper, J. P. F. G.; Ruyter-Spira, C. P.; Kwakman, P. H. S.; Bleeker, W. K.; Keizer, L. C. P.; Bade, J. B.; te Velde, A. A.; Zaat, S. A. J.; Verbeek, M.; Creemers-Molenaar, J.

    2011-01-01

    Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be

  2. Novel nanofibrous dressings containing rhEGF and Aloe vera for wound healing applications.

    Science.gov (United States)

    Garcia-Orue, Itxaso; Gainza, Garazi; Gutierrez, Franciso Borja; Aguirre, Jose Javier; Evora, Carmen; Pedraz, Jose Luis; Hernandez, Rosa Maria; Delgado, Araceli; Igartua, Manoli

    2017-05-25

    Nanofibrous membranes produced by electrospinning possess a large surface area-to-volume ratio, which mimics the three-dimensional structure of the extracellular matrix. Thus, nanofibrous dressings are a promising alternative for chronic wound healing, since they can replace the natural ECM until it is repaired. Therefore, in this study we have developed a PLGA nanofibrous membrane that contains recombinant human Epidermal Growth Factor (rhEGF) and Aloe vera (AV) extract. Both of them promote wound healing, as EGF is a wound healing mediator and AV stimulates the proliferation and activity of fibroblast. The obtained membranes were composed of uniform and randomly oriented fibers with an average diameter of 356.03±112.05nm, they presented a porosity of 87.92±11.96% and the amount of rhEGF was 9.76±1.75μg/mg. The in vitro viability assay demonstrated that the membranes containing rhEGF and AV improved fibroblast proliferation, revealing the beneficial effect of the combination. Furthermore, these membranes accelerated significantly wound closure and reepithelisation in an in vivo full thickness wound healing assay carried out in db/db mice. Overall, these findings demonstrated the potential of PLGA nanofibers containing rhEGF and AV for the treatment of chronic wounds. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Ultrastructure of Sheep Primordial Follicles Cultured in the Presence of Indol Acetic Acid, EGF, and FSH

    Directory of Open Access Journals (Sweden)

    Evelyn Rabelo Andrade

    2011-01-01

    Full Text Available The aim of this study was to investigate the ultrastructural characteristics of primordial follicles after culturing of sheep ovarian cortical slices in the presence of indol acetic acid (IAA, Epidermal Growth Factor (EGF, and FSH. To evaluate ultrastructure of primordial follicles cultured in MEM (control or in MEM containing IAA, EGF, and FSH, fragments of cultured tissue were processes for transmission electron microscopy. Except in the control, primordial follicles cultured in supplemented media for 6 d were ultrastructurally normal. They had oocyte with intact nucleus and the cytoplasm contained heterogeneous-sized lipid droplets and numerous round or elongated mitochondria with intact parallel cristae were observed. Rough endoplasmic reticulum (RER was rarely found. The granulosa cells cytoplasm contained a great number of mitochondria and abundant RER. In conclusion, the presence of IAA, EGF, and FSH helped to maintain ultrastructural integrity of sheep primordial follicles cultured in vitro.

  4. Rescuing apoptotic neurons in Alzheimer’s disease using wheat germ agglutinin-conjugated and cardiolipin-conjugated liposomes with encapsulated nerve growth factor and curcumin

    Directory of Open Access Journals (Sweden)

    Kuo YC

    2015-04-01

    Full Text Available Yung-Chih Kuo, Ching-Chun Lin Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan, Republic of China Abstract: Liposomes with cardiolipin (CL and wheat germ agglutinin (WGA were developed to permeate the blood–brain barrier and treat Alzheimer’s disease. WGA-conjugated and CL-incorporated liposomes (WGA-CL-liposomes were used to transport nerve growth factor (NGF and curcumin (CUR across a monolayer of human brain-microvascular endothelial cells regulated by human astrocytes and to protect SK-N-MC cells against apoptosis induced by ß-amyloid1–42 (Aß1–42 fibrils. An increase in the CL mole percentage in lipids increased the liposomal diameter, absolute zeta potential value, entrapment efficiency of NGF and CUR, release of NGF, biocompatibility, and viability of SK-N-MC cells with Aß1–42, but decreased the atomic ratio of nitrogen to phosphorus and release of CUR. In addition, an increase in the WGA concentration for grafting enhanced the liposomal diameter, atomic ratio of nitrogen to phosphorus, and permeability of NGF and CUR across the blood–brain barrier, but reduced the absolute zeta potential value and biocompatibility. WGA-CL-liposomes carrying NGF and CUR could be promising colloidal delivery carriers for future clinical application in targeting the blood–brain barrier and inhibiting neurotoxicity. Keywords: Alzheimer’s disease, nerve growth factor, curcumin, wheat germ agglutinin, cardiolipin, liposome

  5. Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wen Jen, E-mail: wjlin@ntu.edu.tw; Chien, Wei Hsuan [National Taiwan University, School of Pharmacy, Graduate Institute of Pharmaceutical Sciences (China)

    2015-09-15

    The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly(d,l-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

  6. The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

    International Nuclear Information System (INIS)

    Soula-Rothhut, Mahdhia; Coissard, Cyrille; Sartelet, Herve; Boudot, Cedric; Bellon, Georges; Martiny, Laurent; Rothhut, Bernard

    2005-01-01

    Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF-but not TSP-1-stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development

  7. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    International Nuclear Information System (INIS)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc

    2014-01-01

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors

  8. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc, E-mail: m.muller@ulg.ac.be

    2014-01-15

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.

  9. Immuno-modulatory effect of local rhEGF treatment during tissue repair in diabetic ulcers.

    Science.gov (United States)

    García-Honduvilla, Natalio; Cifuentes, Alberto; Ortega, Miguel A; Pastor, Marta; Gainza, Garazi; Gainza, Eusebio; Buján, Julia; Álvarez-Mon, Melchor

    2018-04-01

    Wound healing is a complex process that can be severely impaired due to pathological situations such as diabetes mellitus. Diabetic foot ulcers are a common complication of this pathology and are characterized by an excessive inflammatory response. In this work, the effects of local treatment with recombinant human epidermal growth factor (rhEGF) were studied using a full-thickness wound healing model in streptozotocin-induced diabetic rats. Wound healing process was assessed with different concentrations of rhEGF (0.1, 0.5, 2.0 and 8.0 µg/mL), placebo and both diabetic and non-diabetic controls ( n  = 53). The macroscopic healing observed in treated diabetic rats was affected by rhEGF concentration. Histologically, we also observed an improvement in the epithelialization, granulation tissue formation and maturation in treated groups, finding again the best response at doses of 0.5 and 2.0 µg/mL. Afterwards, the tissue immune response over time was assessed in diabetic rats using the most effective concentrations of rhEGF (0.5 and 2.0 µg/mL), compared to controls. The presence of macrophages, CD4 + T lymphocytes and CD8 + T lymphocytes, in the reparative tissue was quantified, and cytokine expression was measured by quantitative real-time PCR. rhEGF treatment caused a reduction in the number of infiltrating macrophages in the healing tissue of diabetic, as well as diminished activation of these leukocytes. These findings show that local administration of rhEGF improves the healing process of excisional wounds and the quality of the neoformed tissue in a dose-dependent manner. Besides, this treatment reduces the local inflammation associated with diabetic healing, indicating immuno-modulatory properties. © 2018 The authors.

  10. EGF Prevents the Neuroendocrine Differentiation of LNCaP Cells Induced By Serum Deprivation: The Modulator Role of P13K/Akt

    Directory of Open Access Journals (Sweden)

    Rosa M. Martín-Orozco

    2007-08-01

    Full Text Available The primary focus of this investigation was to study the relationship between neuroendocrine (NE differentiation, epidermal growth factor (EGF because both have been implicated in the progression of prostate cancer. For this purpose, we used gefitinib, trastuzumab, which are inhibitors of EGF receptor (EGFR, ErbB2, respectively. EGF prevents NE differentiation induced by androgen depletion. This effect is prevented by gefitinib, which blocks the activation of EGFR, ErbB2, stimulation of mitogen-activated protein kinase (MAPK, cell proliferation induced by EGF. Conversely, trastuzumab does not inhibit the effect of EGF on EGFR phosphorylation, MAPK activity, cell proliferation, NE differentiation, although it reduces ErbB2 levels specifically, suggesting that ErbB2 is not necessary to inhibit NE differentiation. Prevention of NE differentiation by EGF is mediated by a MAPK-dependent mechanism, requires constitutive Akt activation. The abrogation of the PI3K/Akt pathway changes the role of EGF from inhibitor to inductor of NE differentiation. We show that EGFR tyrosine kinase, MAPK, PI3K inhibitors inhibit the cell proliferation stimulated by EGF but induce the acquisition of NE phenotype. Altogether, the present data should be borne in mind when designing new clinical schedules for the treatment of prostate cancer, including the use of ErbB receptors, associated signaling pathway inhibitors.

  11. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

    International Nuclear Information System (INIS)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui; Chang, Long-Sen

    2016-01-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH 10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50 nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. - Highlights: • Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression. • EGF-induced MMP-9 expression via p300 stabilization and NFκB/c-Jun activation. • Gallic acid

  12. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2016-11-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH 10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50 nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. - Highlights: • Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression. • EGF-induced MMP-9 expression via p300 stabilization and NFκB/c-Jun activation. • Gallic acid

  13. Repeated dose intramuscular injection of the CIMAvax-EGF vaccine in Sprague Dawley rats induces local and systemic toxicity.

    Science.gov (United States)

    Mancebo, A; Casacó, A; González, B; Ledón, N; Sorlozabal, J; León, A; Gómez, D; González, Y; Bada, A M; González, C; Arteaga, M E; Ramírez, H; Fuentes, D

    2012-05-09

    CIMAvax-EGF consists of a human recombinant epidermal growth factor (EGF), coupled to P64k, a recombinant carrier protein from N. meningitis, and Montanide ISA 51 as adjuvant. The vaccine immunization induces a specific antibody production, inhibiting the EGF/EGF-R interaction through EGF deprivation. The objective of this study was to assess the CIMAvax-EGF toxicity in Sprague Dawley rats after intramuscular administration of repeated doses (6 months) and at the same time to determine if rat is a relevant species for studying CIMAvax-EGF vaccine. Rats were randomly distributed into four groups: control, Montanide ISA 51, treated with 1× and 15× of human total dose of the antigen. Animals were immunized weekly during 9 weeks, plus 9 immunizations every 14 days. Rats were inspected daily for clinical signs. Body weight, food consumption, and rectal temperature were measured during the administration of doses. Blood samples were collected for hematological, serum biochemical determinations and EGF titles at the beginning, three months and at the end of experimentation. Gross necropsy and histological examination of tissues were performed on animals at the end of the assay. Vaccine provoked the apparition of antibodies against EGF in the rats, demonstrating rat species relevance in these studies. Body weight gain, food and water consumption were not affected. CIMAvax-EGF and Montanide ISA 51 produced local damage at the administration site, showing multiple cysts and granulomas. Both vaccine-treated groups showed neutrophil elevation, besides an AST increase probably related to the damage at the administration site. Rectal temperature was found to be significantly higher in 15× treated group after immunizations, probably induced by the inflammatory process at the injection site. In summary, the clinical pathology findings together with the body temperature results, appear to be caused by the inflammatory reaction at the administration site of the vaccine, mainly

  14. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    International Nuclear Information System (INIS)

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser 108/121 , HB-EGF-Cys/Ser 116/132 , and HB-EGF-Cys/Ser 134/143 ) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M r of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF 134/143 proteins competed with 125 I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser 134/143 antagonizes EGFRs

  15. Ezrin/NF-kB activation regulates epithelial- mesenchymal transition induced by EGF and promotes metastasis of colorectal cancer.

    Science.gov (United States)

    Li, Yingru; Lin, Zhaoyu; Chen, Bin; Chen, Shuang; Jiang, Zhipeng; Zhou, Taicheng; Hou, Zehui; Wang, Youyuan

    2017-08-01

    There is growing evidence that epithelial mesenchymal-transition (EMT) plays significant roles in terms of tumor metastasis. There are a lot of cytokines inducing EMT of tumor cells, EGF is one of the important cytokines.Ezrin is a connexin between the cytoskeleton and the cell membrane, which is closely related to the morphological movement and metastasis of tumor cells.EGF can activate Ezrin and affects cell motility. In recent years, many studies have shown that NF-kB acts as an important transcription factor, involving in the process of EMT. However, does Ezrin participate in the regulation of EGF-induced EMT through the NF-kB pathway? This question needs us to discuss.In the present study, we found that EGF could induce colorectal cancer cells to develop EMT,enhance their ability to invade and migrate and promotes phosphorylation of Ezrin Tyr353.On the other hand, inhibition of Ezrin could reverse EGF-induced EMT and inhibit NF-kB P65 translocating into the nucleus. Finally, knockout of Ezrin inhibited EGF-induced lung metastasis of colorectal cancer xenografts and abnormal activation of Ezrin and NF-kB were related with colorectal cancer metastasis and poor prognosis. Our present results suggest that Ezrin/NF-kB pathway may provide experimental evidence for new targeted drugs for colorectal cancer metastasis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Renal origin of rat urinary epidermal growth factor

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier

    1984-01-01

    The origin of rat urinary epidermal growth factor (EGF) has been investigated. Unilateral nephrectomy decreased the concentration, total output of EGF and EGF/creatinine ratio by approximately 50%, while the output of creatinine was unchanged. Removal of the submandibular glands and duodenal...... Brunner's glands, organs known to produce EGF, had no influence on the output of EGF in urine. Renal clearance of EGF exceeded that of creatinine, and after bilateral nephrectomy or bilateral ligation of the ureters, the concentration of creatinine in serum increased, while the concentration of EGF...

  17. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  18. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with {sup 99m}Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with {sup 99m}Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 {+-} 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

  19. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99m Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99m Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 ± 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported

  20. Complex Conjugated certificateless-based signcryption with differential integrated factor for secured message communication in mobile network.

    Directory of Open Access Journals (Sweden)

    Sumithra Alagarsamy

    Full Text Available Certificateless-based signcryption overcomes inherent shortcomings in traditional Public Key Infrastructure (PKI and Key Escrow problem. It imparts efficient methods to design PKIs with public verifiability and cipher text authenticity with minimum dependency. As a classic primitive in public key cryptography, signcryption performs validity of cipher text without decryption by combining authentication, confidentiality, public verifiability and cipher text authenticity much more efficiently than the traditional approach. In this paper, we first define a security model for certificateless-based signcryption called, Complex Conjugate Differential Integrated Factor (CC-DIF scheme by introducing complex conjugates through introduction of the security parameter and improving secured message distribution rate. However, both partial private key and secret value changes with respect to time. To overcome this weakness, a new certificateless-based signcryption scheme is proposed by setting the private key through Differential (Diff Equation using an Integration Factor (DiffEIF, minimizing computational cost and communication overhead. The scheme is therefore said to be proven secure (i.e. improving the secured message distributing rate against certificateless access control and signcryption-based scheme. In addition, compared with the three other existing schemes, the CC-DIF scheme has the least computational cost and communication overhead for secured message communication in mobile network.

  1. Complex Conjugated certificateless-based signcryption with differential integrated factor for secured message communication in mobile network.

    Science.gov (United States)

    Alagarsamy, Sumithra; Rajagopalan, S P

    2017-01-01

    Certificateless-based signcryption overcomes inherent shortcomings in traditional Public Key Infrastructure (PKI) and Key Escrow problem. It imparts efficient methods to design PKIs with public verifiability and cipher text authenticity with minimum dependency. As a classic primitive in public key cryptography, signcryption performs validity of cipher text without decryption by combining authentication, confidentiality, public verifiability and cipher text authenticity much more efficiently than the traditional approach. In this paper, we first define a security model for certificateless-based signcryption called, Complex Conjugate Differential Integrated Factor (CC-DIF) scheme by introducing complex conjugates through introduction of the security parameter and improving secured message distribution rate. However, both partial private key and secret value changes with respect to time. To overcome this weakness, a new certificateless-based signcryption scheme is proposed by setting the private key through Differential (Diff) Equation using an Integration Factor (DiffEIF), minimizing computational cost and communication overhead. The scheme is therefore said to be proven secure (i.e. improving the secured message distributing rate) against certificateless access control and signcryption-based scheme. In addition, compared with the three other existing schemes, the CC-DIF scheme has the least computational cost and communication overhead for secured message communication in mobile network.

  2. Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

    Science.gov (United States)

    Chang, Mei-Chi; Chan, Chiu-Po; Chen, Yi-Jane; Hsien, Hsiang-Chi; Chang, Ya-Ching; Yeung, Sin-Yuet; Jeng, Po-Yuan; Cheng, Ru-Hsiu; Hahn, Liang-Jiunn; Jeng, Jiiang-Huei

    2016-03-29

    Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

  3. Identification of potent orally active factor Xa inhibitors based on conjugation strategy and application of predictable fragment recommender system.

    Science.gov (United States)

    Ishihara, Tsukasa; Koga, Yuji; Iwatsuki, Yoshiyuki; Hirayama, Fukushi

    2015-01-15

    Anticoagulant agents have emerged as a promising class of therapeutic drugs for the treatment and prevention of arterial and venous thrombosis. We investigated a series of novel orally active factor Xa inhibitors designed using our previously reported conjugation strategy to boost oral anticoagulant effect. Structural optimization of anthranilamide derivative 3 as a lead compound with installation of phenolic hydroxyl group and extensive exploration of the P1 binding element led to the identification of 5-chloro-N-(5-chloro-2-pyridyl)-3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzamide (33, AS1468240) as a potent factor Xa inhibitor with significant oral anticoagulant activity. We also reported a newly developed Free-Wilson-like fragment recommender system based on the integration of R-group decomposition with collaborative filtering for the structural optimization process. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    Science.gov (United States)

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  5. Aptamer-conjugated gold nanorod for photothermal ablation of epidermal growth factor receptor-overexpressed epithelial cancer

    Science.gov (United States)

    Choi, Jihye; Park, Yeonji; Choi, Eun Bi; Kim, Hyun-Ouk; Kim, Dong Joo; Hong, Yoochan; Ryu, Sung-Ho; Lee, Jung Hwan; Suh, Jin-Suck; Yang, Jaemoon; Huh, Yong-Min; Haam, Seungjoo

    2014-05-01

    Biomarker-specific photothermal nanoparticles that can efficiently sense markers that are overexpressed in distinguished adenocarcinomas have attracted much interest in an aspect of efficacy increase of cancer treatment. We demonstrated a promising prospect of a smart photothermal therapy agent employing anti-epidermal growth factor receptor aptamer (AptEGFR)-conjugated polyethylene glycol (PEG) layted gold nanorods (AptEGFR-PGNRs). The cetyltrimethylammonium bromide bilayer on GNRs was replaced with heterobifunctional PEG (COOH-PEG-SH) not only to serve as a biocompatible stabilizer and but also to conjugate Apt. Subsequently, to direct photothermal therapy agent toward epithelial cancer cells, the carboxylated PEGylated GNRs (PGNRs) were further functionalized with Apt using carbodiimide chemistry. Then, to assess the potential as biomarker-specific photothermal therapy agent of synthesized Apt-PGNRs, the optical properties, biocompatibility, colloidal stability, binding affinity, and epicellial cancer cell killing efficacy in vitro/in vivo under near-infrared laser irradiation were investigated. As a result, Apt-PGNRs exhibit excellent tumor targeting ability and feasibility of effective photothermal ablation cancer therapy.

  6. Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I.

    Science.gov (United States)

    Lim, Chae Jin; Jeon, Jung Eun; Jeong, Se Kyoo; Yoon, Seok Jeong; Kwon, Seon Deok; Lim, Jina; Park, Keedon; Kim, Dae Yong; Ahn, Jeong Keun; Kim, Bong-Woo

    2015-09-01

    Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition.

  7. Epidermal growth factor in the rat prostate

    DEFF Research Database (Denmark)

    Tørring, Niels; Jørgensen, P E; Poulsen, Steen Seier

    1998-01-01

    Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate.......Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate....

  8. Exposure to the cytokine EGF leads to abnormal hyperactivity of pallidal GABA neurons: implications for schizophrenia and its modeling.

    Science.gov (United States)

    Sotoyama, Hidekazu; Namba, Hisaaki; Chiken, Satomi; Nambu, Atsushi; Nawa, Hiroyuki

    2013-08-01

    Previous studies on a cytokine model for schizophrenia reveal that the hyperdopaminergic innervation and neurotransmission in the globus pallidus (GP) is involved in its behavioral impairments. Here, we further explored the physiological consequences of the GP abnormality in the indirect pathway, using the same schizophrenia model established by perinatal exposure to epidermal growth factor (EGF). Single-unit recordings revealed that the neural activity from the lateral GP was elevated in EGF-treated rats in vivo and in vitro (i.e., slice preparations), whereas the central area of the GP exhibited no significant differences. The increase in the pallidal activity was normalized by subchronic treatment with risperidone, which is known to ameliorate their behavioral deficits. We also monitored extracellular GABA concentrations in the substantia nigra, one of the targets of pallidal efferents. There was a significant increase in basal GABA levels in EGF-treated rats, whereas high potassium-evoked GABA effluxes and glutamate levels were not affected. A neurotoxic lesion in the GP of EGF-treated rats normalized GABA concentrations to control levels. Corroborating our in vivo results, GABA release from GP slices was elevated in EGF-treated animals. These findings suggest that the hyperactivity and enhanced GABA release of GP neurons represent the key pathophysiological features of this cytokine-exposure model for schizophrenia. © 2013 International Society for Neurochemistry.

  9. The epidermal growth factor receptor (EGFr) as a target for in situ radiation therapy

    International Nuclear Information System (INIS)

    Vallis, K.A.; Reilly, R.M.

    2003-01-01

    In situ radiation therapy traditionally involves the use of a monoclonal antibody (mAb) directed against a specific tumor-associated antigen and labeled with α-particle emitter such as 131-I. An alternative strategy is to use a low molecular weight peptide rather than a mAb as the carrier molecule. Also, recent evidence shows that radioactive elements that emit Auger electrons may be useful for inducing receptor/cell-specific cytotoxicity. Auger electrons provide low energy emissions (<10-20 keV). Although they have a short range in tissue (a few mm), Auger electrons have a high rate of energy deposition that is comparable to high linear energy transfer radiation such as -particles. Human epidermal growth factor (hEGF) is a natural peptide ligand for EGFr, which is frequently overexpressed in breast cancer. EGF is rapidly internalized and translocated to the cell nucleus following binding to EGFr. We are developing a strategy of EGF conjugated to an Auger electron-emitting radionuclide, 111-In, as a treatment for EGFr-overexpressing breast cancers. This strategy has several advantages over the mAb approach, as EGF is an endogenous peptide and should not be immunogenic. Also, its small molecular size should facilitate extravasation and tumor penetration. We have shown that 111In-hEGF is highly and selectively radiotoxic to MDA-MB-468 human breast cancer cells overexpressing EGFr but was not radiotoxic to MCF-7 breast cancer cells with a 100-fold lower level of EGFr expression. We have also demonstrated that 111-In-hEGF was greater than 80-fold more potent on a molar concentration basis at inhibiting the growth of MDA-MB-468 breast cancer cells than paclitaxel (IC50 70 pM vs. 6 nM respectively) and greater than 400-fold more potent than doxorubicin (IC50 20 nM). We have evaluated the therapeutic efficacy of 111-In-hEGF in athymic mice implanted subcutaneously with MDA-MB-468 breast cancer xenografts. Tumour growth was strongly inhibited following administration of

  10. Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, Krystle J.; Nash, Rebekah P.; Redinbo, Mathew R. (UNC)

    2014-06-16

    The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

  11. Epidermal growth factor in mammary glands and milk from rats

    DEFF Research Database (Denmark)

    Thulesen, J; Raaberg, Lasse; Nexø, Ebba

    1993-01-01

    Epidermal growth factor (EGF) is one of the major growth-promoting agents in milk. Using immunohistochemistry we localized EGF in the mammary glands of lactating rats to the luminal border of the secretory cells. Following proteolytic pretreatment of the histological sections, the EGF-immunoreact......Epidermal growth factor (EGF) is one of the major growth-promoting agents in milk. Using immunohistochemistry we localized EGF in the mammary glands of lactating rats to the luminal border of the secretory cells. Following proteolytic pretreatment of the histological sections, the EGF...

  12. Characterization of a new CAMP factor carried by an integrative and conjugative element in Streptococcus agalactiae and spreading in Streptococci.

    Directory of Open Access Journals (Sweden)

    Sarah Chuzeville

    Full Text Available Genetic exchanges between Streptococci occur frequently and contribute to their genome diversification. Most of sequenced streptococcal genomes carry multiple mobile genetic elements including Integrative and Conjugative Elements (ICEs that play a major role in these horizontal gene transfers. In addition to genes involved in their mobility and regulation, ICEs also carry genes that can confer selective advantages to bacteria. Numerous elements have been described in S. agalactiae especially those integrated at the 3' end of a tRNA(Lys encoding gene. In strain 515 of S. agalactiae, an invasive neonate human pathogen, the ICE (called 515_tRNA(Lys is functional and carries different putative virulence genes including one encoding a putative new CAMP factor in addition to the one previously described. This work demonstrated the functionality of this CAMP factor (CAMP factor II in Lactococcus lactis but also in pathogenic strains of veterinary origin. The search for co-hemolytic factors in a collection of field strains revealed their presence in S. uberis, S. dysgalactiae, but also for the first time in S. equisimilis and S. bovis. Sequencing of these genes revealed the prevalence of a species-specific factor in S. uberis strains (Uberis factor and the presence of a CAMP factor II encoding gene in S. bovis and S. equisimilis. Furthermore, most of the CAMP factor II positive strains also carried an element integrated in the tRNA(Lys gene. This work thus describes a CAMP factor that is carried by a mobile genetic element and has spread to different streptococcal species.

  13. Does epidermal growth factor play a role in the action of sucralfate?

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier

    1987-01-01

    Epidermal growth factor (EGF) is a mitogenic peptide synthesized in the submandibular glands and released in saliva. EGF is able to prevent the development of gastrointestinal ulcers in the rat and to accelerate their healing. The present work was undertaken to examine whether Sucralfate acts via...... the effector system of EGF. We conclude that Sucralfate does not change the binding of EGF to its receptor, but it is able to bind EGF in a pH dependent manner and at pH below 4.5 virtually all EGF is bound to Sucralfate. In vivo studies in rats with acid-induced gastric ulcers show that sucralfate carries EGF...... to the ulcer, and that EGF is available for a longer period of time (3 hours) when EGF and Sucralfate are is given together than when EGF is given alone....

  14. Study of the effects of low-dose radiation and rhEGF on growth of cultured human epithelial cells

    International Nuclear Information System (INIS)

    Yang Jicheng; Zhao Xiaoyu; Sheng Weihua; Tang Zhongyi

    1998-01-01

    In authors' study, the method of taking skin sample, mincing and trypsinizing the sample are presented. The cells were inoculated on adherent membrane or, for sublethally injured 3T3 cells, in culture dish fed with Eargles' medium supplemented with fetal calf serum and various growth-stimulating factors. The cultures were incubated at 37 degree C in an atmosphere containing 5% CO 2 . The medium was changed every three days. The cultured cells became confluent in about two weeks. At the same time, low-dose-radiation and rhEGF were used to influence the growth of the epithelial cells and to test the effects of dosage and concentration. The results showed that low-dose-radiation in the conditions like authors' study could enhance the growth of human epithelial cells just like rhEGF, and it has synergetic effects with rhEGF. The mechanism is discussed

  15. Tc-99m direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    International Nuclear Information System (INIS)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto

    2005-01-01

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  16. La ciclooxigenasa-2 (COX-2 y el factor de crecimiento epidérmico (EFG en lesiones epiteliales orales premalignas Cyclooxygenase-2 (COX-2 and epidermal growth factor (EGF in oral premalignant epithelial lesions

    Directory of Open Access Journals (Sweden)

    S. Díaz Prado

    2009-06-01

    Full Text Available Las lesiones premalignas orales incluyen eritroplasias (manchas rojas y leucoplasias (manchas blancas, las cuales se desarrollan a lo largo de superficies epiteliales. Estas lesiones son considerados marcadores en la "carcinogénesis de campo" ya que pacientes con lesiones premalignas orales pueden desarrollar carcinoma de células escamosas (CCS en el sitio de las lesiones, así como en otros lugares de tracto aerodigestivo superior. Se está haciendo un gran esfuerzo para identificar nuevos biomarcadores SEBs (surrogate endpoint biomarkers para el carcinoma de células escamosas de cabeza y cuello. Los SEBs candidatos para el carcinoma de células escamosas invasivo en el trato aerodigestivo superior deben ser detectables con los cambios moleculares celulares y tisulares que tienen lugar durante la formación del tumor. Entre los diferentes marcadores que se han propuesto hasta la actualidad, la ciclooxigenasa- 2 (COX-2 y el receptor del factor de crecimiento epidérmico (EGFR parecen ser los más prometedores. COX-2 se sobre expresa durante el proceso tumoral, desde hiperplasia temprana a enfermedad metastásica. EGFR también está anormalmente activado en tumores epiteliales, pues las células de casi todas estas neoplasias expresan altos niveles de este receptor, una característica asociada con un peor pronóstico clínico. En este sentido el tracto aerodigestivo superior proporciona un sistema o modelo único para el estudio de CCS y para la investigación de nuevos candidatos SEBs.Oral premalignant lesions include leukoplakia (white patch and erythroplakia (red patch, which develop on epithelial surfaces. These lesions are markers for field cancerization because patients with oral premalignancy can develop squamous cell carcinoma at the site of the lesion(s and at other sites in the upper aerodigestive tract. An effort is being made to identify surrogate endpoint biomarkers (SEBs for head and neck squamous cell carcinoma (HNSCC

  17. Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition

    DEFF Research Database (Denmark)

    Feng, Yongjia; Demehri, Farok R; Xiao, Weidong

    2017-01-01

    BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal...... deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfr(knock out (KO)) and IEC-pik3r1(KO) mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC...

  18. EGF does not induce Msx-1 and Msx-2 in dental mesenchyme.

    Science.gov (United States)

    Wang, Y H; Kollar, E J; Upholt, W B; Mina, M

    1998-01-01

    Previous heterospecific tissue recombinations indicate that mandibular epithelium exerts the first known inductive signal for odontogenesis in mouse embryos. BMP-4 and EGF are two growth factors implicated as signaling molecules mediating the initial inductive epithelial-mesenchymal interactions during odontogenesis. The purpose of the present study was to examine and compare the effects of these growth factors and mouse mandibular epithelium on expression of Msx-1 and Msx-2 genes in molar-forming mesenchyme. Agarose beads soaked in growth factors or pieces of mouse mandibular epithelium (E11) were placed in contact with E11 molar-forming mesenchyme and cultured for 24 h. Whole-mount in situ hybridization analysis revealed that, in contrast to mouse mandibular epithelium and BMP-4-releasing beads, EGF-releasing beads did not induce the expression of Msx-1 and Msx-2 in E11 molar-forming mesenchyme. These observations suggest that whereas BMP-4 may be involved in activation of Msx-1 and Msx-2 in the underlying mesenchyme, EGF may regulate events involved in the formation of dental lamina.

  19. Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

    Science.gov (United States)

    Gong, Wei; Dou, Huan; Liu, Xianqin; Sun, Lingyun; Hou, Yayi

    2012-10-01

    1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 μg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1β. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.

  20. Advanced computational biology methods identify molecular switches for malignancy in an EGF mouse model of liver cancer.

    Directory of Open Access Journals (Sweden)

    Philip Stegmaier

    Full Text Available The molecular causes by which the epidermal growth factor receptor tyrosine kinase induces malignant transformation are largely unknown. To better understand EGFs' transforming capacity whole genome scans were applied to a transgenic mouse model of liver cancer and subjected to advanced methods of computational analysis to construct de novo gene regulatory networks based on a combination of sequence analysis and entrained graph-topological algorithms. Here we identified transcription factors, processes, key nodes and molecules to connect as yet unknown interacting partners at the level of protein-DNA interaction. Many of those could be confirmed by electromobility band shift assay at recognition sites of gene specific promoters and by western blotting of nuclear proteins. A novel cellular regulatory circuitry could therefore be proposed that connects cell cycle regulated genes with components of the EGF signaling pathway. Promoter analysis of differentially expressed genes suggested the majority of regulated transcription factors to display specificity to either the pre-tumor or the tumor state. Subsequent search for signal transduction key nodes upstream of the identified transcription factors and their targets suggested the insulin-like growth factor pathway to render the tumor cells independent of EGF receptor activity. Notably, expression of IGF2 in addition to many components of this pathway was highly upregulated in tumors. Together, we propose a switch in autocrine signaling to foster tumor growth that was initially triggered by EGF and demonstrate the knowledge gain form promoter analysis combined with upstream key node identification.

  1. Microfluidic generated EGF-gradients induce chemokinesis of transplantable retinal progenitor cells via the JAK/STAT and PI3kinase signaling pathways.

    Directory of Open Access Journals (Sweden)

    Uchenna J Unachukwu

    Full Text Available A growing number of studies are evaluating retinal progenitor cell (RPC transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR and evaluated whether exposure to epidermal growth factor (EGF can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400 ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.

  2. Gene Expression of the EGF System-a Prognostic Model in Non-Small Cell Lung Cancer Patients Without Activating EGFR Mutations

    DEFF Research Database (Denmark)

    Sandfeld-Paulsen, Birgitte; Folkersen, Birgitte Holst; Rasmussen, Torben Riis

    2016-01-01

    OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non-small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands is a l.......17-6.47], P model that takes the complexity of the EGF system into account and shows that this model is a strong prognostic marker in NSCLC patients.......OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non-small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands...... is a likely explanation. The aim of this study is to demonstrate that the combined network of receptors and ligands from the EGF system is a prognostic marker. MATERIAL AND METHODS: Gene expression of the receptors EGFR, HER2, HER3, HER4, and the ligands AREG, HB-EGF, EPI, TGF-α, and EGF was measured...

  3. Ultrastructure of sheep primordial follicles cultured in the presence of indol acetic acid, EGF, and FSH

    DEFF Research Database (Denmark)

    Andrade, Evelyn Rabelo; Hyttel, Poul; Landim-Alvarenga, Fernanda Da Cruz

    2011-01-01

    The aim of this study was to investigate the ultrastructural characteristics of primordial follicles after culturing of sheep ovarian cortical slices in the presence of indol acetic acid (IAA), Epidermal Growth Factor (EGF), and FSH. To evaluate ultrastructure of primordial follicles cultured...... in MEM (control) or in MEM containing IAA, EGF, and FSH, fragments of cultured tissue were processes for transmission electron microscopy. Except in the control, primordial follicles cultured in supplemented media for 6¿d were ultrastructurally normal. They had oocyte with intact nucleus...... and the cytoplasm contained heterogeneous-sized lipid droplets and numerous round or elongated mitochondria with intact parallel cristae were observed. Rough endoplasmic reticulum (RER) was rarely found. The granulosa cells cytoplasm contained a great number of mitochondria and abundant RER. In conclusion...

  4. SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

    Science.gov (United States)

    Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

    2016-10-25

    Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

  5. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

    Science.gov (United States)

    Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

    2005-11-21

    It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

  6. Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

    Science.gov (United States)

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

    2016-06-01

    Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

  7. EGF-induced expansion of migratory cells in the rostral migratory stream.

    Directory of Open Access Journals (Sweden)

    Olle R Lindberg

    Full Text Available The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF. In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin(+/Olig2(+ cells in the RMS. Negative for NG2 and CNPase, these radixin(+/Olig2(+ cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2(+ population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin(+/Olig2(+ progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.

  8. Long-term organ culture of rabbit skin: Effect of EGF on epidermal structure in vitro

    International Nuclear Information System (INIS)

    Kondo, S.; Hozumi, Y.; Aso, K.

    1990-01-01

    A method is described for maintaining the epidermal structure of normal rabbit ear skin explants in organ culture for up to 12 weeks. Split-thickness skin specimens were put in diffusion chambers made of either millipore filters or bovine collagen membranes, and then submitted to a roller tube culture at 15 rpm and 36 degrees C. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) + 0.4 micrograms/ml hydrocortisone. The gas used in the culture tube was air + 5% CO2. Autoradiography revealed the incorporation of [3H]-glycine into the 68-kD keratin band of explants for up to 12 weeks, indicating that normal keratinization was maintained throughout the entire culture period. The turnover time of the epidermis from basal layer to granular layer was around 7 d in both the early and late stages of culture. The addition of epidermal growth factor (EGF) to the culture caused the epidermis to become acanthotic with orthokeratosis, but with high concentrations of EGF (greater than or equal to 10 ng/ml) parakeratosis and increased proliferation of the epidermis occurred. Dexamethasone (DMS) strongly inhibited the EGF effect

  9. A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis.

    Science.gov (United States)

    Philippar, Ulrike; Roussos, Evanthia T; Oser, Matthew; Yamaguchi, Hideki; Kim, Hyung-Do; Giampieri, Silvia; Wang, Yarong; Goswami, Sumanta; Wyckoff, Jeffrey B; Lauffenburger, Douglas A; Sahai, Erik; Condeelis, John S; Gertler, Frank B

    2008-12-01

    The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.

  10. Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

    DEFF Research Database (Denmark)

    Lambert, S.; Ameels, H.; Gniadecki, R.

    2008-01-01

    The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internal......The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo...... internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests...... the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption Udgivelsesdato: 2008/12...

  11. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui; Chang, Long-Sen

    2016-11-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles

    Science.gov (United States)

    Chen, Lijue; She, Xiaodong; Wang, Tao; He, Li; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

    2015-08-01

    Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The effect and mechanism of 5-FU loaded EGF grafted HMSNs (EGF-HMSNs-5-FU) in overcoming acquired drug resistance in SW480/ADR cells were studied. The EGF-HMSNs were demonstrated to be specifically internalized in EGFR overexpressed SW480/ADR cells via a receptor-mediated endocytosis and can escape from endo-lysosomes. The EGF-HMSNs-5-FU exhibited much higher cytotoxicity on SW480/ADR cells than HMSNs-5-FU and free 5-FU while the plain HMSNs did not show significant cytotoxicity. The mechanism of EGF-HMSNs-5-FU in overcoming drug resistance in SW480/ADR cells could be attributed to the specific internalization of EGF-HMSNs-5-FU in EGFR overexpressed cells which can lead to high intracellular drug accumulation and cause cell death through S phase arrest.Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The

  13. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  14. Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yun-Jeong; Cho, Hyun-Ji [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Magae, Junji [Magae Bioscience Institute, 49-4 Fujimidai, Tsukuba 300-1263 (Japan); Lee, In-Kyu [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Park, Keun-Gyu, E-mail: kpark@knu.ac.kr [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2013-12-15

    Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.

  15. In vitro and in vivo imaging of prostate cancer angiogenesis using anti-vascular endothelial growth factor receptor 2 antibody-conjugated quantum dot

    International Nuclear Information System (INIS)

    Kwon, Haejin; Lee, Jiyeon; Song, Rita; Lee, Jung Han; Hwang, Sung Il; Lee, Hak Jong; Kim, Young Hwa

    2013-01-01

    Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.

  16. The effects of EGF and IGF-1 on FSH-mediated in vitro maturation of domestic cat oocytes derived from follicular and luteal stages.

    Science.gov (United States)

    Yıldırım, Koray; Vural, M Rıfat; Küplülü, Sükrü; Ozcan, Ziya; Polat, I Mert

    2014-04-01

    The objective of this study was to evaluate the influence of epidermal growth factor (EGF) and insulin like growth factor-I (IGF-1) on the in vitro maturation of cat oocytes recovered from follicular and luteal stage ovaries. Oocytes from follicular (n=580) and luteal (n=209) stages were harvested and divided into four groups, which were cultured in FSH-mediated maturation medium supplemented with: (1) EGF alone (25ng/mL); (2) IGF-1 alone (100ng/mL); (3) EGF+IGF-1 (25ng/mL EGF+100ng/mL IGF-I); or (4) no growth factor (control). The proportion of follicular stage oocytes reaching the metaphase II stage was significantly higher than that of oocytes obtained at the luteal stage in both control and study groups (pIGF-1, and 78.1% in EGF+IGF-1 groups, whereas the respective values for gametes collected from luteal stage ovaries were 12.5%, 17.5%, 12.5%, and 16.9%. Additionally, the differences between the study and control groups were significant in the case of follicular stage oocytes. Finally, supplementing the maturation medium with EGF and/or IGF-1 significantly enhanced the meiotic maturation of oocytes recovered from follicular stage ovaries. The present study also demonstrated that the combination of EGF and IGF-I provides an additional or synergic effect on meiotic maturation of oocytes recovered from the follicular stage. Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  17. Immunohistochemical localization of epidermal growth factor in rat and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1986-01-01

    Epidermal growth factor (EGF) is a peptide which stimulates cell mitotic activity and differentiation, has a cytoprotective effect on the gastroduodenal mucosa, and inhibits gastric acid secretion. The immunohistochemical localization of EGF in the Brunner's glands and the submandibular glands is...... antisera against human urinary EGF worked in rat as well as man. EGF was found only in cells with an exocrine function.......Epidermal growth factor (EGF) is a peptide which stimulates cell mitotic activity and differentiation, has a cytoprotective effect on the gastroduodenal mucosa, and inhibits gastric acid secretion. The immunohistochemical localization of EGF in the Brunner's glands and the submandibular glands...... is well documented. The localization of EGF in other tissues is still unclarified. In the present study, the immunohistochemical localization of EGF in tissues from rat, man and a 20 week human fetus were investigated. In man and rat, immunoreaction was found in the submandibular glands, the serous glands...

  18. ICESag37, a Novel Integrative and Conjugative Element Carrying Antimicrobial Resistance Genes and Potential Virulence Factors in Streptococcus agalactiae.

    Science.gov (United States)

    Zhou, Kaixin; Xie, Lianyan; Han, Lizhong; Guo, Xiaokui; Wang, Yong; Sun, Jingyong

    2017-01-01

    ICE Sag37 , a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors, was characterized in a clinical isolate of Streptococcus agalactiae . Two clinical strains of S. agalactiae , Sag37 and Sag158, were isolated from blood samples of new-borns with bacteremia. Sag37 was highly resistant to erythromycin and tetracycline, and susceptible to levofloxacin and penicillin, while Sag158 was resistant to tetracycline and levofloxacin, and susceptible to erythromycin. Transfer experiments were performed and selection was carried out with suitable antibiotic concentrations. Through mating experiments, the erythromycin resistance gene was found to be transferable from Sag37 to Sag158. Sma I-PFGE revealed a new Sma I fragment, confirming the transfer of the fragment containing the erythromycin resistance gene. Whole genome sequencing and sequence analysis revealed a mobile element, ICE Sag37 , which was characterized using several molecular methods and in silico analyses. ICE Sag37 was excised to generate a covalent circular intermediate, which was transferable to S. agalactiae . Inverse PCR was performed to detect the circular form. A serine family integrase mediated its chromosomal integration into rumA , which is a known hotspot for the integration of streptococcal ICEs. The integration site was confirmed using PCR. ICE Sag37 carried genes for resistance to multiple antibiotics, including erythromycin [ erm(B) ], tetracycline [ tet(O) ], and aminoglycosides [ aadE, aphA , and ant(6) ]. Potential virulence factors, including a two-component signal transduction system ( nisK/nisR ), were also observed in ICE Sag37 . S1-PFGE analysis ruled out the existence of plasmids. ICE Sag37 is the first ICE Sa2603 family-like element identified in S. agalactiae carrying both resistance and potential virulence determinants. It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S. agalactiae .

  19. Cell model for the study of receptor and regulatory functions of human proHB-EGF

    Directory of Open Access Journals (Sweden)

    N. V. Korotkevych

    2014-08-01

    Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

  20. Human epidermal growth factor: molecular forms and application of radioimmunoassay and radioreceptor assay

    International Nuclear Information System (INIS)

    Hirata, Y.; Orth, D.N.

    1981-01-01

    Epidermal growth factor (EGF), a 53 amino acid polypeptide, was first isolated by Cohen. EGF's growth-promoting activity is not limited to epidermal cells, but is expressed on a wide variety of tissues derived from a number of different species. Human EGF (hEGF) was isolated and subsequently purified from human urine. Unexpectedly, a close structural relationship was recognized between mEGF and human β-urogastrone. The authors recently developed both an homologous hEGF radioimmunoassay (RIA) and a radioreceptor assay (RRA) using a human placental membrane fraction. Using these assays, the molecular size of hEGF in human body fluids and tissues was evaluated, and partial characterization of a high molecular weight form of hEGF isolated from human urine was carried out. The concentrations of immunoreactive hEGF were also determined in human tissues and plasma after extraction either with cationic exchange chromatography or with immunoaffinity chromatography. (Auth.)

  1. SNT-2 interacts with ERK2 and negatively regulates ERK2 signaling in response to EGF stimulation

    International Nuclear Information System (INIS)

    Huang Lin; Gotoh, Noriko; Zhang Shengliang; Shibuya, Masabumi; Yamamoto, Tadashi; Tsuchida, Nobuo

    2004-01-01

    The control of cellular responses with fibroblast growth factors and neurotrophins is mediated through membrane-linked docking proteins, SNT (suc1-binding neurotrophic target)-1/FRS2α and SNT-2/FRS2β. ERK1/2 are members of the mitogen-activated protein kinase family that regulate diverse cellular activities in response to various stimuli. Here, we demonstrate that SNT-2 does not become tyrosine phosphorylated significantly in response to EGF but forms a complex with ERK2 via the region of 186-252 amino acid residues, and the complex formation is enhanced upon EGF stimulation. SNT-2 downregulates ERK2 phosphorylation, suppresses and delays ERK2 nuclear accumulation which occurs following EGF stimulation. In contrast, the mutant SNT-2 which carries deletion of 186-252 amino acids and lacks ERK2 binding does not have these effects. These observations suggest that SNT-2 negatively regulates ERK2 signaling activated via EGF stimulation through direct binding to ERK2

  2. An immunologic approach to induction of epidermal growth factor deficiency

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1995-01-01

    Epidermal growth factor (EGF) in pharmacologic doses is able to induce growth and development in the fetus and the newborn. To investigate the opposite situation, the effects of insufficient amounts of EGF during development, we wanted to establish an in vivo model with a state of EGF deficiency....

  3. Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma.

    Science.gov (United States)

    Tang, Jiaze; Huang, Ning; Zhang, Xiang; Zhou, Tao; Tan, Ying; Pi, Jiangli; Pi, Li; Cheng, Si; Zheng, Huzhi; Cheng, Yuan

    2017-01-01

    The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD)-labeled aptamer (QD-Apt) nanoprobe by conjugating aptamer 32 (A32) to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs) were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling glioma cell lines and human brain glioma tissues, and target gliomas in situ was also investigated. We found that not only could QD-Apt specially bind to the U87-EGFRvIII glioma cells but also bind to human glioma tissues in vitro. Fluorescence imaging in vivo with orthotopic glioma model mice bearing U87-EGFRvIII showed that QD-Apt could penetrate the blood-brain barrier and then selectively accumulate in the tumors through binding to EGFRvIII, and consequently, generate a strong fluorescence, which contributed to the margins of gliomas that were visualized clearly, and thus, help the surgeons realize the maximum safe resection of glioma. In addition, QD-Apt can also be applied in preoperative diagnosis and postoperative examination of glioma

  4. Chronic treatment with epidermal growth factor causes esophageal epithelial hyperplasia in pigs and rats

    DEFF Research Database (Denmark)

    Juhl, C O; Vinter-Jensen, Lars; Poulsen, Steen Seier

    1995-01-01

    Epidermal growth factor (EGF) is an important factor for maintaining the esophageal functional integrity. Goettingen minipigs were treated with either placebo or subcutaneous EGF (30 micrograms/kg/day) for four weeks. Wistar rats were treated with either placebo or subcutaneous EGF (150 microgram...

  5. Systemic treatment with epidermal growth factor in pigs induces ductal proliferations in the pancreas

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Juhl, C O; Teglbjaerg, P S

    1997-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and the EGF receptor are often overexpressed in chronic pancreatitis and in malignant pancreatic growth. Transgenic mice overexpressing TGF-alpha develop tissue changes in the pancrease resembling changes found in chronic...... pancreatitis. The effects of systemic treatment with EGF on the porcine pancrease were investigated in this study....

  6. Genetic variations in EGF and EGFR and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, Sara; Andersson, Ulrika; Liu, Yanhong

    2010-01-01

    associated EGF polymorphisms in haplotype block 4 were validated in a set from MDACC; however, none of the associations were clearly replicated. rs379644 had a hazard ratio (HR) of 1.19 (0.94-1.51) in the whole population with 18.6 months survival in the risk genotype compared with 24.5 in the reference...

  7. A mouse model of otitis media identifies HB-EGF as a mediator of inflammation-induced mucosal proliferation.

    Directory of Open Access Journals (Sweden)

    Keigo Suzukawa

    Full Text Available Otitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae.From a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors.From the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes.Our data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.

  8. The safety and efficacy of EGF-based cream for the prevention of radiotherapy-induced skin injury: results from a multicenter observational study

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Hyun Cheol [Dept.of Radiation Oncology, Seoul National University College of Medicine, Seoul (Korea, Republic of); Ahn, Seung Do [Dept.of Radiation Oncology, Asan Medical Center, Seoul (Korea, Republic of); Choi, Doo Ho [Dept.of Radiation Oncology, Samsung Medical Center, Seoul (Korea, Republic of); Kang, Min Kyu [Dept.of Radiation Oncology, eungnam University College of Medicine, Daegu (Korea, Republic of); Wu, Hong Gyun [Dept.of Radiation Oncology, Chonnam National University Hwasun Hospital, Huasun (Korea, Republic of)

    2014-09-15

    This study was designed to evaluate the efficacy and safety of topically applied recombinant human epidermal growth factor (rhEGF) for the prevention of radiation-induced dermatitis in cancer patients. From December 2010 to April 2012, a total of 1,172 cancer patients who received radiotherapy (RT) of more than 50 Gy were prospectively enrolled and treated with EGF-based cream. An acute skin reaction classified according to the Radiation Therapy Oncology Group 6-point rating scale was the primary end point and we also assessed the occurrence of edema, dry skin, or pruritus. The percentage of radiation dermatitis with maximum grade 0 and grade 1 was 19% and 58% at the time of 50 Gy, and it became 29% and 47% after completion of planned RT. This increment was observed only in breast cancer patients (from 18%/62% to 32%/49%). Adverse events related to the EGF-based cream developed in 49 patients (4%) with mild erythema the most common. Skin toxicity grade >2 was observed in 5% of the patients. Edema, dry skin, and pruritus grade > or =3 developed in 9%, 9%, and 1% of the patients, respectively. Prophylactic use of an EGF-based cream is effective in preventing radiation dermatitis with tolerable toxicity. Further studies comparing EGF cream with other topical agents may be necessary.

  9. EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells.

    Science.gov (United States)

    Mergler, Stefan; Pleyer, Uwe; Reinach, Peter; Bednarz, Jürgen; Dannowski, Haike; Engelmann, Katrin; Hartmann, Christian; Yousif, Tarik

    2005-02-01

    Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.

  10. The safety and efficacy of EGF-based cream for the prevention of radiotherapy-induced skin injury: results from a multicenter observational study

    International Nuclear Information System (INIS)

    Kang, Hyun Cheol; Ahn, Seung Do; Choi, Doo Ho; Kang, Min Kyu; Wu, Hong Gyun

    2014-01-01

    This study was designed to evaluate the efficacy and safety of topically applied recombinant human epidermal growth factor (rhEGF) for the prevention of radiation-induced dermatitis in cancer patients. From December 2010 to April 2012, a total of 1,172 cancer patients who received radiotherapy (RT) of more than 50 Gy were prospectively enrolled and treated with EGF-based cream. An acute skin reaction classified according to the Radiation Therapy Oncology Group 6-point rating scale was the primary end point and we also assessed the occurrence of edema, dry skin, or pruritus. The percentage of radiation dermatitis with maximum grade 0 and grade 1 was 19% and 58% at the time of 50 Gy, and it became 29% and 47% after completion of planned RT. This increment was observed only in breast cancer patients (from 18%/62% to 32%/49%). Adverse events related to the EGF-based cream developed in 49 patients (4%) with mild erythema the most common. Skin toxicity grade >2 was observed in 5% of the patients. Edema, dry skin, and pruritus grade > or =3 developed in 9%, 9%, and 1% of the patients, respectively. Prophylactic use of an EGF-based cream is effective in preventing radiation dermatitis with tolerable toxicity. Further studies comparing EGF cream with other topical agents may be necessary.

  11. The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier

    1991-01-01

    shows that the membrane fraction of homogenized rat kidney contains an enzyme that releases immuno and receptor reactive EGF from the kidney membranes when incubated at 37 degrees C. Gel filtration shows that the EGF reactivity released from the membranes is similar to the EGF reactivity in rat urine......High levels of epidermal growth factor (EGF) are excreted in the urine and high levels of mRNA for the EGF-precursor have been demonstrated in the kidney. The EGF-precursor is a membrane bound peptide in the kidney, but little is known about the renal processing of the precursor. The present study...

  12. Hair growth is promoted by BeauTop via expression of EGF and FGF‑7.

    Science.gov (United States)

    Lee, Chien-Ying; Yang, Chi-Yu; Lin, Ching-Che; Yu, Min-Chien; Sheu, Shuenn-Jyi; Kuan, Yu-Hsiang

    2018-06-01

    Minoxidil and finasteride have been approved to treat hair loss by the Food and Drug Administration. However, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. BeauTop (BT) is a health food supplement which contains Ginseng radix, Astragali radix, Radix Angelicae sinensis, Ligustri fructus, Rehmannia glutinosa and Eclipta prostrata (Linn). Susbsequent to oral administration of BT at 0.6 g/kg/day to wax/rosin‑induced alopecia in C57BL/6 mice, BT significantly induced hair growth at day 8 compared with control treatment (P<0.05). The expression levels of epidermal growth factor (EGF), and fibroblast growth factor (FGF)‑7 were increased compared with control animals on day 8. In contrast, levels of FGF‑5 of the BT group were reduced compared with the control on day 12. There were no effects on the expression of insulin‑like growth factor 1. The results demonstrated that the mechanism of BT improving alopecia is potentially associated with modulation of EGF and FGF‑7 levels. Taken together, it is suggested that BT may have a potential effect of the promotion of hair growth.

  13. Cetuximab-conjugated nanodiamonds drug delivery system for enhanced targeting therapy and 3D Raman imaging.

    Science.gov (United States)

    Li, Dandan; Chen, Xin; Wang, Hong; Liu, Jie; Zheng, Meiling; Fu, Yang; Yu, Yuan; Zhi, Jinfang

    2017-12-01

    In this study, a multicomponent nanodiamonds (NDs)-based targeting drug delivery system, cetuximab-NDs-cisplatin bioconjugate, combining both specific targeting and enhanced therapeutic efficacy capabilities, is developed and characterized. The specific targeting ability of cetuximab-NDs-cisplatin system on human liver hepatocellular carcinoma (HepG2) cells is evaluated through epidermal growth factor receptor (EGFR) blocking experiments, since EGFR is over-expressed on HepG2 cell membrane. Besides, cytotoxic evaluation confirms that cetuximab-NDs-cisplatin system could significantly inhibit the growth of HepG2 cells, and the therapeutic activity of this system is proven to be better than that of both nonspecific NDs-cisplatin conjugate and specific EGF-NDs-cisplatin conjugate. Furthermore, a 3-dimensional (3D) Raman imaging technique is utilized to visualize the targeting efficacy and enhanced internalization of cetuximab-NDs-cisplatin system in HepG2 cells, using the NDs existing in the bioconjugate as Raman probes, based on the characteristic Raman signal of NDs at 1332 cm -1 . These advantageous properties of cetuximab-NDs-cisplatin system propose a prospective imaging and treatment tool for further diagnostic and therapeutic purposes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Chronic administration of epidermal growth factor to pigs induces growth, especially of the urinary tract with accumulation of epithelial glycoconjugates

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Juhl, C O; Poulsen, Steen Seier

    1995-01-01

    Epidermal growth factor (EGF) receptor hyperstimulation induced by systemically administered EGF or by the development of transgenic mice overexpressing transforming growth factor alpha (TGF alpha) or other EGF-related ligands is known to induce various effects, such as acceleration of developmen...

  15. Tc-{sup 99m} direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: crdias@ipen.br

    2005-10-15

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  16. Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma

    Directory of Open Access Journals (Sweden)

    Tang J

    2017-05-01

    Full Text Available Jiaze Tang,1 Ning Huang,1 Xiang Zhang,1,2 Tao Zhou,3 Ying Tan,1,4 Jiangli Pi,5 Li Pi,1 Si Cheng,6 Huzhi Zheng,5 Yuan Cheng1 1Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, 2Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging, 3Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, 4Institute of Life Sciences, Chongqing Medical University, 5Key Laboratory on Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, 6Department of Orthopaedics, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China Abstract: The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD-labeled aptamer (QD-Apt nanoprobe by conjugating aptamer 32 (A32 to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling

  17. Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

    NARCIS (Netherlands)

    van Belzen, N.; Spaargaren, M.; Verkleij, A. J.; Boonstra, J.

    1990-01-01

    Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction,

  18. Expression of epidermal growth factor receptors in human endometrial carcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent...

  19. Bacteriophytochromes control conjugation in Agrobacterium fabrum.

    Science.gov (United States)

    Bai, Yingnan; Rottwinkel, Gregor; Feng, Juan; Liu, Yiyao; Lamparter, Tilman

    2016-08-01

    Bacterial conjugation, the transfer of single stranded plasmid DNA from donor to recipient cell, is mediated through the type IV secretion system. We performed conjugation assays using a transmissible artificial plasmid as reporter. With this assay, conjugation in Agrobacterium fabrum was modulated by the phytochromes Agp1 and Agp2, photoreceptors that are most sensitive in the red region of visible light. In conjugation studies with wild-type donor cells carrying a pBIN-GUSINT plasmid as reporter that lacked the Ti (tumor inducing) plasmid, no conjugation was observed. When either agp1(-) or agp2(-) knockout donor strains were used, plasmid DNA was delivered to the recipient, indicating that both phytochromes suppress conjugation in the wild type donor. In the recipient strains, the loss of Agp1 or Agp2 led to diminished conjugation. When wild type cells with Ti plasmid and pBIN-GUS reporter plasmid were used as donor, a high rate of conjugation was observed. The DNA transfer was down regulated by red or far-red light by a factor of 3.5. With agp1(-) or agp2(-) knockout donor cells, conjugation in the dark was about 10 times lower than with the wild type donor, and with the double knockout donor no conjugation was observed. These results imply that the phytochrome system has evolved to inhibit conjugation in the light. The decrease of conjugation under different temperature correlated with the decrease of phytochrome autophosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Adrenergic effects on renal secretion of epidermal growth factor in the rat

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1985-01-01

    Urinary epidermal growth factor (EGF) has been demonstrated recently to originate from the kidneys. The present study was undertaken to investigate the adrenergic and cholinergic influence on secretion of renal EGF. beta-Adrenergic agonists increased the level of urinary EGF, while propranolol......, a beta-adrenergic blocking agent, decreased basal and beta-adrenergic stimulated total output of urinary EGF. Acetylcholine and the anticholinergic agent atropine had no effect on the output of EGF in urine. Also chemical sympathectomy induced by 6-hydroxydopamine reduced the urinary output of EGF. None...... of the experimental groups had a median serum concentration above the detection limit of the assay. The present study shows that secretion of renal EGF is under the influence of the sympathetic nervous system and release of EGF is stimulated by activation of beta-adrenergic receptors in the kidneys....

  1. Epidermal growth factor-mediated effects on equine vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Grosenbaugh, D.A.; Amoss, M.S.; Hood, D.M.; Morgan, S.J.; Williams, J.D.

    1988-01-01

    Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125 I-labeled EGF, indicate the presence of a single class of high-affinity binding sites, with an estimated maximal binding capacity of 5,800 sites/cells. EGF stimulated [ 3 H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 x 10 -11 M. Likewise, EGF-mediated cellular proliferation was dose dependent under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease

  2. Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

    International Nuclear Information System (INIS)

    Ogiwara, Kazutaka; Nagaoka, Masato; Cho, Chong-Su; Akaike, Toshihiro

    2006-01-01

    We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg 2+ although integrin-mediated cell adhesion to natural ECMs is dependent on Mg 2+ . Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF

  3. Fetal antigen 1, an EGF multidomain protein in the sex hormone-producing cells of the gonads and the microenvironment of germ cells

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Erb, K; Westergaard, L G

    1999-01-01

    Fetal antigen 1 (FA1), an epidermal growth factor (EGF) multidomain glycoprotein, was investigated in the human reproductive system. Immunohistochemical analysis of the male reproductive system revealed staining for FA1 in the Leydig cells only. Concentrations of FA1 in seminal plasma and serum w...

  4. Combined immunotherapy of breast cancer with EGF and VEGF vaccines from DNA shuffling in a mouse model.

    Science.gov (United States)

    Jin, Dong; Yu, Xin; Chen, Bing; Li, Zhitao; Ding, Jia; Zhao, Xiuyun; Qi, Gaofu

    2017-06-01

    Development of EGF and VEGF vaccines with high antigenicity for combined immunotherapy of EGF-EGFR signaling-dependent epithelial tumors such as breast cancer. EGF genes from mouse, human and chicken were randomly assembled to chimeric genes by DNA shuffling, then a chimeric EGF was selected out by PCR, SDS-PAGE and immunization for combined immunotherapy of breast cancer with a previously constructed chimeric VEGF vaccine from shuffling. Combined vaccination with chimeric EGF and VEGF from shuffling could induce high titer of antibodies against EGF and VEGF to inhibit tumor growth and angiogenesis, and improve the survival rate of mice with breast cancer. Combined vaccination with EGF and VEGF from shuffling showed better immunotherapy on EGF-EGFR signaling-dependent epithelial tumors such as breast cancer than the single-agent EGF vaccination.

  5. Magnetism of an Excited Self-Conjugate Nucleus: Precise Measurement of the g Factor of the 21+ State in Mg24

    CERN Document Server

    Kusoglu, A; Georgiev, G; Brown, B  A; Goasduff, A; Atanasova, L; Balabanski, D  L; Bostan, M; Danchev, M; Detistov, P; Gladnishki, K  A; Ljungvall, J; Matea, I; Radeck, D; Sotty, C; Stefan, I; Verney, D; Yordanov, D  T

    2015-01-01

    A precise measurement of the g factor of the first-excited state in the self-conjugate (N=Z) nucleus Mg24 is performed by a new time-differential recoil-in-vacuum method based on the hyperfine field of hydrogenlike ions. Theory predicts that the g factors of such states, in which protons and neutrons occupy the same orbits, should depart from 0.5 by a few percent due to configuration mixing and meson-exchange effects. The experimental result, g=0.538±0.013, is in excellent agreement with recent shell-model calculations and shows a departure from 0.5 by almost 3 standard deviations, thus achieving, for the first time, the precision and accuracy needed to test theory. Proof of the new method opens the way for wide applications including measurements of the magnetism of excited states of exotic nuclei produced as radioactive beams.

  6. dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

    International Nuclear Information System (INIS)

    Baladron, Victoriano; Ruiz-Hidalgo, Maria Jose; Nueda, Maria Luisa; Diaz-Guerra, Maria Jose M.; Garcia-Ramirez, Jose Javier; Bonvini, Ezio; Gubina, Elena; Laborda, Jorge

    2005-01-01

    The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis

  7. Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence.

    Science.gov (United States)

    Strand, Marie; Micchelli, Craig A

    2013-01-01

    Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.

  8. Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence.

    Directory of Open Access Journals (Sweden)

    Marie Strand

    Full Text Available Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs of the acidic copper cell region (CCR, which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.

  9. An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion.

    Science.gov (United States)

    Hu, Xuefeng; Neoh, Koon-Gee; Shi, Zhilong; Kang, En-Tang; Poh, Chyekhoon; Wang, Wilson

    2010-12-01

    The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. An effective approach to minimize implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time. In this in vitro study, the surfaces of titanium (Ti) substrates were functionalized by first covalently grafting either dopamine followed by carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Vascular endothelial growth factor (VEGF) was then conjugated to the polysaccharide-grafted surface. Antibacterial assay with Staphylococcus aureus (S. aureus) showed that the polysaccharide-modified substrates significantly decrease bacterial adhesion. The CMCS-functionalized Ti demonstrated better antibacterial property than the HAC-functionalized Ti since CMCS is bactericidal while HA only inhibits the adhesion of bacteria without killing them. Osteoblast attachment, as well as alkaline phosphatase (ALP) activity and calcium deposition were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Thus, Ti substrates modified with polysaccharides conjugated with VEGF can promote osteoblast functions and concurrently reduce bacterial adhesion. Since VEGF is also known to enhance angiogenesis, the VEGF-polysaccharide functionalized substrates will have promising applications in the orthopedic field. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Elevated EGF Levels in the Blood Serum of Dogs with Periodontal Diseases and Oral Tumours.

    Science.gov (United States)

    Sobczyńska-Rak, Aleksandra; Żylińska, Beata; Polkowska, Izabela; Szponder, Tomasz

    2018-01-01

    Paradontopathy and neoplasms of the oral cavity represent one of the greatest challenges in human and animal dentistry. EGF plays a key role in maintaining the integrity and proper rate of cell proliferation in normal oral epithelium. The aim of the present study was to study serum levels of EGF in dogs diagnosed with periodontal diseases and oral cavity tumours. The samples comprised of cancerous tissue sections and serum obtained from dogs of various breeds, aged between 5-13 years. Serum EGF concentrations were measured by an immunoenzymatic method. The median for EGF concentration in serum of dogs suffered from severe periodontal diseases was greater when compared to the control group. EGF concentration in dogs with malignant tumours was significantly higher than in those with non-malignant growths. A positive correlation between EGF concentration and tumour size was also observed. EGF level in dogs diagnosed with benign tumours was comparable to the control group. The blood serum level of EGF increases significantly in patients with malignant oral tumours and advanced periodontal disease. In malignant tumours, the high level of EGF correlates with the size and invasiveness of the neoplasm. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  11. Epidermal growth factor and its receptors in human pancreatic carcinoma

    International Nuclear Information System (INIS)

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N.

    1990-01-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells

  12. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

    Science.gov (United States)

    Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

    1993-05-06

    Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

  13. The effect of epidermal growth factor and IGF-I infusion on hepatic and renal expression of the IGF-system in adult female rats

    NARCIS (Netherlands)

    J.W. van Neck (Han); E.M. Berghout; L. Vinter-Jensen; C.A.H. Groffen; V. Cingel-Ristic; N.F. Dits (Natasja); S.L.S. Drop (Stenvert); A. Flyvbjerg (Allan)

    2000-01-01

    textabstractSystemic administration of epidermal growth factor (EGF) in neonatal rats results in reduced body weight gain and decreased circulating levels of IGF-I, suggesting its involvement in EGF-induced growth retardation. We investigated the effect of EGF and/or IGF-I

  14. Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

    1999-08-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

  15. Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

    1999-01-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

  16. TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells.

    Directory of Open Access Journals (Sweden)

    Zhi-Ren Zhang

    Full Text Available Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+ or in which cilia are absent (cilia (-. This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (- compared to cilia (+ cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (- but not cilia (+ cells. In addition EGF increased cell proliferation in cilia (- cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium.We conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

  17. EGF-R is Expressed and AP-1 and NF-κ:B Are Activated in Stromal Myofibroblasts Surrounding Colon Adenocarcinomas Paralleling Expression of COX-2 and VEGF

    Directory of Open Access Journals (Sweden)

    Panagiotis A. Konstantinopoulos

    2007-01-01

    Full Text Available Background: COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-κ:B transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-κ:B, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN, p-Iκ:B-α (phosphorylated Iκ:B-α, EGF-R, COX-2, NF-κ:B and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p-Iκ:B-α, NF-κ:B, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-Iκ:B-α, NF-κ:B, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-κ:B transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production.

  18. Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

    Science.gov (United States)

    Cheng, Simon K; Olale, Felix; Brivanlou, Ali H

    2004-01-01

    Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity. PMID:14966532

  19. Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors.

    Directory of Open Access Journals (Sweden)

    Simon K Cheng

    2004-02-01

    Full Text Available Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.

  20. Overexpression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor Mediates Liver Fibrosis in Transgenic Mice.

    Science.gov (United States)

    Guo, Yongze; Ding, Qian; Chen, Lei; Ji, Chenguang; Hao, Huiyao; Wang, Jia; Qi, Wei; Xie, Xiaoli; Ma, Junji; Li, Aidi; Jiang, Xiaoyu; Li, Xiaotian; Jiang, Huiqing

    2017-08-01

    The role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver fibrosis is not clear and is sometimes even contradictory. To clarify this role, a HB-EGF transgenic (Tg) mouse model was, for the first time, used to evaluate the functions of HB-EGF in liver fibrosis. For the in vivo study, carbon tetrachloride injection and bile duct ligation treatment were used to induce liver fibrosis in HB-EGF Tg mice and wild-type (WT) mice, respectively. Primary hepatic satellite cells (HSCs) were isolated from HB-EGF Tg and WT mice for the in vitro study. Compared with the WT mice, HB-EGF Tg mice were shown to develop more severe liver fibrosis when treated with carbon tetrachloride or bile duct ligation, with increased matrix metalloproteinases 13 activity and enhanced expression of fibrogenic genes including α-smooth muscle actin and collagen I. HB-EGF gene transfer led to an increase in proliferation and a decrease in apoptosis in primary HSCs. The ERK signaling pathway was more highly activated in primary HSCs from HB-EGF Tg mice than in those from WT mice. Our investigation confirmed the profibrotic effect of HB-EGF on the liver using a Tg mouse model. This result may contribute to the elucidation of HB-EGF as a therapeutic target in liver fibrosis. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

  1. Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1989-01-01

    Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell

  2. Urinary transforming growth factors in neoplasia: separation of 125I-labeled transforming growth factor-alpha from epidermal growth factor in human urine

    International Nuclear Information System (INIS)

    Stromberg, K.; Hudgins, W.R.

    1986-01-01

    Purified human epidermal growth factor (hEGF) from urine promotes anchorage-independent cell growth in soft agar medium. This growth is enhanced by transforming growth factor-beta (TGF-beta), and is specifically inhibited by hEGF antiserum. Transforming growth factors of the alpha type (TGF-alpha), potentially present in normal human urine or urine from tumor-bearing patients, also promote anchorage-independent cell growth and compete with EGF for membrane receptor binding. Consequently, TGF-alpha cannot be distinguished from urinary hEGF by these two functional assays. Therefore, a technique for separation of TGF-alpha and related peptides from urinary EGF based on biochemical characteristics would be useful. Radioiodination of characterized growth factors [mouse EGF (mEGF), hEGF, and rat TGF-alpha (rTGF-alpha)], which were then separately added to human urine, was used to evaluate a resolution scheme that separates TGF-alpha from the high level of background hEGF present in human urine. Methyl bonded microparticulate silica efficiently adsorbed the 125 I-labeled mEGF, 125 I-labeled hEGF, and 125 I-labeled rTGF-alpha that were added to 24-h human urine samples. Fractional elution with acetonitrile (MeCN) of the adsorbed silica released approximately 70 to 80% of the 125 I-labeled mEGF and 125 I-labeled hEGF between 25 and 30% MeCN, and over 80% of the 125 I-labeled rTGF-alpha between 15 and 25% MeCN, with retention after dialysis of less than 0.2 and 1.7% of the original urinary protein, respectively. A single-step enrichment of about 400-fold for mEGF and hEGF, and 50-fold for rTGF-alpha were achieved rapidly. 125 I-labeled mEGF and 125 I-labeled hEGF eluted later than would be predicted on the basis of their reported molecular weight of approximately 6000, whereas 125 I-labeled rTGF-alpha eluted from Bio-Gel P-10 at an approximate molecular weight of 8000 to 9000

  3. The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

    Science.gov (United States)

    Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun

    2016-11-01

    Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  4. Adrenergic effects on secretion of epidermal growth factor from Brunner's glands

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1985-01-01

    The influence of the sympathetic nervous system and adrenergic agonists on flow rate and secretion of epidermal growth factor (EGF) from Brunner's glands has been investigated in the rat. Chemical sympathectomy by administration of 6-hydroxydopamine increased volume secretion and output of EGF from...... Brunner's glands but depleted the glands of EGF. Infusion of noradrenaline, an alpha-adrenergic agonist, inhibited basal and vasoactive intestinal polypeptide (VIP) stimulated flow rate and output of EGF from Brunner's glands and increased the amount of EGF in the tissue. Vasoactive intestinal polypeptide...... also increased the amount of EGF in Brunner's gland tissue and this was unchanged after simultaneous infusion of VIP and noradrenaline as well as VIP and isoproterenol, a beta-adrenergic agonist. Isoproterenol had no effect on basal and VIP stimulated secretion of EGF from Brunner's glands...

  5. Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1984-01-01

    The present study was undertaken to investigate the effect of alpha- and beta-adrenergic agonists on secretion of epidermal growth factor (EGF) from the rat submandibular glands and to test the possibility of intestinal absorption of EGF. Alpha-adrenergic agonists increased the concentration...... of salivary EGF by approximately a hundred times, while the serum concentration of EGF was unchanged. The contents of EGF in the submandibular glands decreased upon administration of the alpha-adrenergic agonist noradrenaline, and this was confirmed on immunohistochemical investigation of the glands. Beta-adrenergic....... This study shows that alpha-adrenergic agonists stimulate exocrine secretion of submandibular EGF and that EGF in physiological amounts are not absorbed in the gastrointestinal tract....

  6. Self-assembly behaviours of peptide-drug conjugates: influence of multiple factors on aggregate morphology and potential self-assembly mechanism

    Science.gov (United States)

    Fan, Qin; Ji, Yujie; Wang, Jingjing; Wu, Li; Li, Weidong; Chen, Rui; Chen, Zhipeng

    2018-04-01

    Peptide-drug conjugates (PDCs) as self-assembly prodrugs have the unique and specific features to build one-component nanomedicines. Supramolecular structure based on PDCs could form various morphologies ranging from nanotube, nanofibre, nanobelt to hydrogel. However, the assembly process of PDCs is too complex to predict or control. Herein, we investigated the effects of extrinsic factors on assembly morphology and the possible formation of nanostructures based on PDCs. To this end, we designed a PDC consisting of hydrophobic drug (S)-ketoprofen (Ket) and valine-glutamic acid dimeric repeats peptide (L-VEVE) to study their assembly behaviour. Our results showed that the critical assembly concentration of Ket-L-VEVE was 0.32 mM in water to form various nanostructures which experienced from micelle, nanorod, nanofibre to nanoribbon. The morphology was influenced by multiple factors including molecular design, assembly time, pH and hydrogen bond inhibitor. On the basis of experimental results, we speculated the possible assembly mechanism of Ket-L-VEVE. The π-π stacking interaction between Ket molecules could serve as an anchor, and hydrogen bonded-induced β-sheets and hydrophilic/hydrophobic balance between L-VEVE peptide play structure-directing role in forming filament-like or nanoribbon morphology. This work provides a new sight to rationally design and precisely control the nanostructure of PDCs based on aromatic fragment.

  7. Brain Injury Expands the Numbers of Neural Stem Cells and Progenitors in the SVZ by Enhancing Their Responsiveness to EGF

    Directory of Open Access Journals (Sweden)

    Dhivyaa Alagappan

    2009-04-01

    Full Text Available There is an increase in the numbers of neural precursors in the SVZ (subventricular zone after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.

  8. Autocrine production of TGF-β confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    International Nuclear Information System (INIS)

    Castillo, Gaelle del; Murillo, Miguel M.; Alvarez-Barrientos, Alberto; Bertran, Esther; Fernandez, Margarita; Sanchez, Aranzazu; Fabregat, Isabel

    2006-01-01

    Transforming growth factor-beta (TGF-β) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-β-treated fetal hepatocytes: TβT-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes TβT-FH to die after serum withdrawal. TβT-FH expresses high levels of transforming growth factor-alpha (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-α antibody restores the capacity of cells to die. TGF-β, which is expressed by TβT-FH, mediates up-regulation of TGF-α and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-β confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands

  9. Insulin induces a transcriptional activation of epiregulin, HB-EGF and amphiregulin, by a PI3K-dependent mechanism: Identification of a specific insulin-responsive promoter element

    International Nuclear Information System (INIS)

    Ornskov, Dorthe; Nexo, Ebba; Sorensen, Boe S.

    2007-01-01

    Previously we have shown that insulin-stimulation of RT4 bladder cancer cells leads to increased proliferation, which require HER1 activation, and is accompanied by increased mRNA expression of the EGF-ligands heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), and epiregulin (EPI) [D. Ornskov, E. Nexo, B.S. Sorensen, Insulin-induced proliferation of bladder cancer cells is mediated through activation of the epidermal growth factor system, FEBS J. 273 (2006) 5479-5489]. In the present paper, we have investigated the molecular mechanism leading to this insulin-induced expression. We monitored the decay of mRNA after inhibiting transcription with Actinomycin D and demonstrated that the insulin-mediated increase was not caused by enhanced mRNA stability. In untreated cells, HB-EGF mRNA was the least stable, whereas AR and EPI mRNA decayed with slower kinetics. However, promoter analysis of HB-EGF and EPI demonstrated that insulin stimulated transcription. Studies on the EPI promoter identified the insulin-responsive element to be located in the region -564 to -365 bp. This region contains potential binding sites for the transcription factors SP1, AP1, and NF-κB. Interestingly, all three transcription factors can be activated by PI3K. We demonstrate that the insulin-induced expression of HB-EGF, AR, and EPI mRNA is completely prevented by the specific PI3K inhibitor Wortmannin, suggesting an involvement of the PI3K

  10. Transforming growth factor alpha and epidermal growth factor in laryngeal carcinomas demonstrated by immunohistochemistry

    DEFF Research Database (Denmark)

    Christensen, M E; Therkildsen, M H; Poulsen, Steen Seier

    1993-01-01

    the basal cell layer. The present investigation and our previous results confirm the existence of EGF receptors, TGF-alpha and EGF in laryngeal carcinomas. In addition, we conclude that the conditions do exist for growth factors to act through an autocrine system in poorly differentiated tumours and through......Fifteen laryngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) using immunohistochemical methods. In a recent study the same material was characterized for epidermal growth factor receptors (EGF...... receptors) which were confined predominantly to the undifferentiated cells. The expression of this growth factor system in malignant cells may play a role in carcinogenesis and/or tumour growth. All carcinomas were positive for TGF-alpha and 12 were positive for EGF. In moderately-to-well differentiated...

  11. Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

    Science.gov (United States)

    Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

    2015-01-01

    E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

  12. Epidermal growth factor treatment decreases mortality and is associated with improved gut integrity in sepsis.

    Science.gov (United States)

    Clark, Jessica A; Clark, Andrew T; Hotchkiss, Richard S; Buchman, Timothy G; Coopersmith, Craig M

    2008-07-01

    Epidermal growth factor (EGF) is a cytoprotective peptide that has healing effects on the intestinal mucosa. We sought to determine whether systemic administration of EGF after the onset of sepsis improved intestinal integrity and decreased mortality. FVB/N mice were subjected to either sham laparotomy or 2 x 23 cecal ligation and puncture (CLP). Septic mice were further randomized to receive injection of either 150 microg kg(-1) d(-1) (i.p.) EGF or 0.9% saline (i.p.). Circulating EGF levels were decreased after CLP compared with sham animals but were unaffected by giving exogenous EGF treatment. In contrast, intestinal EGF levels increased after CLP and were further augmented by exogenous EGF treatment. Intestinal EGF receptor was increased after CLP, whether assayed by immunohistochemistry, real-time polymerase chain reaction, or Western blot, and exogenous EGF treatment decreased intestinal EGF receptor. Villus length decreased 2-fold between sham and septic animals, and EGF treatment resulted in near total restitution of villus length. Sepsis decreased intestinal proliferation and increased intestinal apoptosis. This was accompanied by increased expression of the proapoptotic proteins Bid and Fas-associated death domain, as well as the cyclin-dependent kinase inhibitor p21 cip1/waf Epidermal growth factor treatment after the onset of sepsis restored both proliferation and apoptosis to levels seen in sham animals and normalized expression of Bid, Fas-associated death domain, and p21 cip1/waf . To determine whether improvements in gut homeostasis were associated with a decrease in sepsis-induced mortality, septic mice with or without EGF treatment after CLP were followed 7 days for survival. Mortality decreased from 60% to 30% in mice treated with EGF after the onset of sepsis (P < 0.05). Thus, EGF may be a potential therapeutic agent for the treatment of sepsis in part due to its ability to protect intestinal integrity.

  13. Decline in Proliferation and Immature Neuron Markers in the Human Subependymal Zone during Aging: Relationship to EGF- and FGF-related Transcripts

    Directory of Open Access Journals (Sweden)

    Christin Weissleder

    2016-11-01

    Full Text Available Neuroblasts exist within the human subependymal zone (SEZ; however, it is debated to what extent neurogenesis changes during normal aging. It is also unknown how precursor proliferation may correlate with the generation of neuronal and glial cells or how expression of growth factors and receptors may change throughout the adult lifespan. We provided evidence of dividing cells in the human SEZ in conjunction with a dramatic age-related decline (n=50; 21-103 years of mRNAs indicative of proliferating cells (Ki67 and immature neurons (doublecortin. Microglia mRNA (ionized calcium-binding adapter molecule 1 increased during aging, whereas transcript levels of stem/precursor cells (glial fibrillary acidic protein delta and achaete-scute homolog 1, astrocytes (vimentin and glial fibrillary acidic protein and oligodendrocytes (oligodendrocyte lineage transcription factor 2 remained stable. Epidermal growth factor receptor (EGFR and fibroblast growth factor 2 (FGF2 mRNAs increased throughout adulthood, while transforming growth factor alpha (TGFα, EGF, Erb-B2 receptor tyrosine kinase 4 (ErbB4 and FGF receptor 1 (FGFR1 mRNAs were unchanged across adulthood. Cell proliferation mRNA positively correlated with FGFR1 transcripts. Immature neuron and oligodendrocyte expression positively correlated with TGFα and ErbB4 mRNAs, whilst astrocyte transcripts positively correlated with EGF, FGF2 and FGFR1 mRNAs. Microglia mRNA positively correlated with EGF and FGF2 expression. Our findings indicate that neurogenesis in the human SEZ continues well into adulthood, although proliferation and neuronal differentiation may decline across adulthood. We suggest that mRNA expression of EGF- and FGF-related family members do not become limited during aging and may modulate neuronal and glial fate determination in the SEZ throughout human life.

  14. Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

    Science.gov (United States)

    Fu, Xiang-Dong

    2017-04-01

    EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

  15. In brown adipocytes, adrenergically induced β1-/β3-(Gs)-, α2-(Gi)- and α1-(Gq)-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    International Nuclear Information System (INIS)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-01-01

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α 1 -adrenoceptor coupled via G q ), clonidine (α 2 via G i ) or CL316243 (β 3 via G s ) or via β 1 -receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC 50 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR-induced Erk1/2 activation. •

  16. Conjugation in Escherichia coli

    Science.gov (United States)

    Boyer, Herbert

    1966-01-01

    Boyer, Herbert (Yale University, New Haven, Conn.). Conjugation in Escherichia coli. J. Bacteriol. 91:1767–1772. 1966.—The sex factor of Escherichia coli K-12 was introduced into an E. coli B/r strain by circumventing the host-controlled modification and restriction incompatibilities known to exist between these closely related strains. The sexual properties of the constructed F+ B strain and its Hfr derivatives were examined. These studies showed that the E. coli strain B/r F+ and Hfr derivatives are similar to the E. coli strain K-12 F+ and Hfr derivatives. However, the site of sex factor integration was found to be dependent on the host genome. PMID:5327905

  17. Comparative measurement of ghrelin, leptin, adiponectin, EGF and IGF-1 in breast milk of mothers with overweight/obese and normal-weight infants.

    Science.gov (United States)

    Khodabakhshi, A; Ghayour-Mobarhan, M; Rooki, H; Vakili, R; Hashemy, S-I; Mirhafez, S R; Shakeri, M-T; Kashanifar, R; Pourbafarani, R; Mirzaei, H; Dahri, M; Mazidi, M; Ferns, G; Safarian, M

    2015-05-01

    Obese infants are more susceptible to develop adulthood obesity and its related comorbidities. Previous studies have shown the presence of hormones and growth factors in maternal breast milk that may influence infant adiposity. The aim of this study was to investigate differences in concentrations of three hormones and two growth factors in the breast milk of mothers with obese and non-obese infants. In this cross-sectional study, 40 mothers with overweight or obese infants (weight for length percentile >97) and 40 age-matched mothers with normal-weight infant (-10 milk concentrations of ghrelin and adiponectin, leptin, epithelial growth factor (EGF) and insulin-like growth factor-1 (IGF-1) were measured using enzyme-linked immunosorbent assay methods. The mean breast milk concentration of ghrelin was higher in mothers with normal-weight infants, 137.50 pg/ml, than in mothers with obese infants, 132.00 pg/ml (P=0.001). This was also true regarding the concentration of EGF in mothers with (0/04 ng/ml) and without (0/038 ng/ml) normal-weight infants (P=0.01). No significant differences were observed in concentrations of leptin, adiponectin and IGF-1 between two groups (P > 0.05). There was also a significant positive correlation between EGF and ghrelin in both groups. This study revealed that there was a correlation between ghrelin and EGF level in breast milk of mothers with obese and non-obese infants, suggesting a possible regulatory effect of these two hormones on weight in infants.

  18. Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

    Directory of Open Access Journals (Sweden)

    Emilia Galperin

    Full Text Available Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2. To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF receptor (EGFR, we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

  19. Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-01-01

    of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10...... with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P

  20. Renal content and output of epidermal growth factor in long-term adrenergic agonist-treated rats

    DEFF Research Database (Denmark)

    Thulesen, J; Nexø, Ebba; Poulsen, Steen Seier

    2000-01-01

    This study investigates the renal and urinary levels of epidermal growth factor (EGF) in rats under long-term treatment with alpha- or beta-adrenergic agonists. Urine samples were obtained on days 7, 14 and 21, and renal tissue samples on day 21. EGF was quantified by ELISA and tissue sections were...... material in the distal tubules. Concomitantly, reduced levels of EGF and EGF mRNA were observed, and also the urinary levels of EGF were reduced. Together, these observations indicate alpha-adrenergic treatment to affect the distal tubules. Treatment with the beta-adrenergic agonist did not change...... fractional kidney weight, but initially the urinary excretion of EGF was reduced. The data add further evidence to the suggestion that activity of the sympathetic nervous system influences renal homeostasis of EGF, either directly or indirectly through renal histopathological changes....

  1. Urine Epidermal Growth Factor, Monocyte Chemoattractant Protein-1 or Their Ratio as Biomarkers for Interstitial Fibrosis and Tubular Atrophy in Primary Glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Supanat Worawichawong

    2016-12-01

    Full Text Available Background/Aims: The degree of tubular atrophy and interstitial fibrosis (IFTA is an important prognostic factor in glomerulonephritis. Imbalance between pro-inflammatory cytokines such as monocyte chemoattractant protein- 1 (MCP-1 and protective cytokines such as epidermal growth factor (EGF likely determine IFTA severity. In separate studies, elevated MCP-1 and decreased EGF have been shown to be associated with IFTA severity. In this study, we aim to evaluate the predictive value of urinary EGF/MCP-1 ratio compared to each biomarker individually for moderate to severe IFTA in primary glomerulonephritis (GN. Methods: Urine samples were collected at biopsy from primary GN (IgA nephropathy, focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy. MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assay. Results: EGF, MCP-1 and EGF/MCP-1 ratio from primary GN, all correlated with IFTA (n=58. By univariate analysis, glomerular filtration rate, EGF, and EGF/MCP-1 ratio were associated with IFTA. By multivariate analysis, only EGF/MCP-1 ratio was independently associated with IFTA. EGF/MCP-1 ratio had a sensitivity of 88% and specificity of 74 % for IFTA. EGF/MCP-1 had good discrimination for IFTA (AUC=0.85, but the improvement over EGF alone was not significant. Conclusion: EGF/MCP-1 ratio is independently associated IFTA severity in primary glomerulonephritis, but the ability of EGF/MCP-1 ratio to discriminate moderate to severe IFTA may not be much better than EGF alone.

  2. Cytotoxic T lymphocyte-dependent tumor growth inhibition by a vascular endothelial growth factor-superantigen conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Qingwen [Shanghai Chest Hospital, Shanghai 200433 (China); State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433 (China); Jiang, Songmin [State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433 (China); Han, Baohui [Shanghai Chest Hospital, Shanghai 200433 (China); Sun, Tongwen [Wuhan Junyu Innovation Pharmaceuticals, Inc., Wuhan 430079 (China); Li, Zhengnan; Zhao, Lina; Gao, Qiang [College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Sun, Jialin, E-mail: jialin_sun@126.com [Wuhan Junyu Innovation Pharmaceuticals, Inc., Wuhan 430079 (China)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer We construct and purify a fusion protein VEGF-SEA. Black-Right-Pointing-Pointer VEGF-SEA strongly repressed the growth of murine solid sarcoma 180 (S180) tumors. Black-Right-Pointing-Pointer T cells driven by VEGF-SEA were accumulated around tumor cells bearing VEGFR by mice image model. Black-Right-Pointing-Pointer VEGF-SEA can serve as a tumor targeting agent and sequester CTLs into the tumor site. Black-Right-Pointing-Pointer The induced CTLs could release the cytokines, perforins and granzyme B to kill the tumor cells. -- Abstract: T cells are major lymphocytes in the blood and passengers across the tumor vasculature. If these T cells are retained in the tumor site, a therapeutic potential will be gained by turning them into tumor-reactive cytotoxic T lymphocytes (CTLs). A fusion protein composed of human vascular endothelial growth factor (VEGF) and staphylococcal enterotoxin A (SEA) with a D227A mutation strongly repressed the growth of murine solid sarcoma 180 (S180) tumors (control versus VEGF-SEA treated with 15 {mu}g, mean tumor weight: 1.128 g versus 0.252 g, difference = 0.876 g). CD4{sup +} and CD8{sup +} T cells driven by VEGF-SEA were accumulated around VEGFR expressing tumor cells and the induced CTLs could release the tumoricidal cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Meanwhile, intratumoral CTLs secreted cytolytic pore-forming perforin and granzyme B proteins around tumor cells, leading to the death of tumor cells. The labeled fusion proteins were gradually targeted to the tumor site in an imaging mice model. These results show that VEGF-SEA can serve as a tumor targeting agent and sequester active infiltrating CTLs into the tumor site to kill tumor cells, and could therefore be a potential therapeutical drug for a variety of cancers.

  3. Epidermal Growth Factor and Intestinal Barrier Function

    Directory of Open Access Journals (Sweden)

    Xiaopeng Tang

    2016-01-01

    Full Text Available Epidermal growth factor (EGF is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health.

  4. A histological and micro-CT investigation in to the effect of NGF and EGF on the periodontal, alveolar bone, root and pulpal healing of replanted molars in a rat model - a pilot study.

    Science.gov (United States)

    Furfaro, Francesco; Ang, Estabelle S M; Lareu, Ricky R; Murray, Kevin; Goonewardene, Mithran

    2014-01-06

    This study aims to investigate, utilising micro-computed tomography (micro-CT) and histology, whether the topical application of nerve growth factor (NGF) and/or epidermal growth factor (EGF) can enhance periodontal, alveolar bone, root and pulpal tissue regeneration while minimising the risk of pulpal necrosis, root resorption and ankylosis of replanted molars in a rat model. Twelve four-week-old male Sprague-Dawley rats were divided into four groups: sham, collagen, EGF and NGF. The maxillary right first molar was elevated and replanted with or without a collagen membrane impregnated with either the growth factors EGF or NGF, or a saline solution. Four weeks after replantation, the animals were sacrificed and the posterior maxilla was assessed using histological and micro-CT analysis. The maxillary left first molar served as the control for the corresponding right first molar. Micro-CT analysis revealed a tendency for all replanted molars to have reduced root length, root volume, alveolar bone height and inter-radicular alveolar bone volume. It appears that the use of the collagen membrane had a negative effect while no positive effect was noted with the incorporation of EGF or NGF. Histologically, the incorporation of the collagen membrane was found to negatively affect pulpal, root, periodontal and alveolar bone healing with pulpal inflammation and hard tissue formation, extensive root resorption and alveolar bone fragmentation. The incorporation of EGF and NGF did not improve root, periodontal or alveolar bone healing. However, EGF was found to improve pulp vascularisation while NGF-improved pulpal architecture and cell organisation, although not to the level of the control group. Results indicate a possible benefit on pulpal vascularisation and pulpal cell organisation following the incorporation of EGF and NGF, respectively, into the alveolar socket of replanted molars in the rat model. No potential benefit of EGF and NGF was detected in periodontal or root

  5. Investigation of Epidermal Growth Factor, Tumor Necrosis Factor-alpha and Thioredoxin System in Rats Exposed to Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Erol-Demirbilek Melike

    2016-09-01

    Full Text Available Background: Thioredoxin reductase (TrxR, epidermal growth factor (EGF and tumor necrosis factor-α (TNF-α have neuroprotective/neurotoxic effects in cerebral ischemia. We aimed to investigate the TrxR activity, EGF and TNF-α levels in cerebral ischemic, sham-operated and non-ischemic rat brains.

  6. Recombinant epidermal growth factor-like domain-1 from coagulation factor VII functionalized iron oxide nanoparticles for targeted glioma magnetic resonance imaging.

    Science.gov (United States)

    Liu, Heng; Chen, Xiao; Xue, Wei; Chu, Chengchao; Liu, Yu; Tong, Haipeng; Du, Xuesong; Xie, Tian; Liu, Gang; Zhang, Weiguo

    The highly infiltrative and invasive nature of glioma cells often leads to blurred tumor margins, resulting in incomplete tumor resection and tumor recurrence. Accurate detection and precise delineation of glioma help in preoperative delineation, surgical planning and survival prediction. In this study, recombinant epidermal growth factor-like domain-1, derived from human coagulation factor VII, was conjugated to iron oxide nanoparticles (IONPs) for targeted glioma magnetic resonance (MR) imaging. The synthesized EGF1-EGFP-IONPs exhibited excellent targeting ability toward tissue factor (TF)-positive U87MG cells and human umbilical vein endothelial cells in vitro, and demonstrated persistent and efficient MR contrast enhancement up to 12 h for preclinical glioma models with high targeting specificity in vivo. They hold great potential for clinical translation and developing targeted theranostics against brain glioma.

  7. EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

    Directory of Open Access Journals (Sweden)

    Har-Vardi Iris

    2007-01-01

    Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

  8. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    International Nuclear Information System (INIS)

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-01-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [ 125 I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  9. Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

    Science.gov (United States)

    Stidl, R; Fuchs, S; Bossard, M; Siekmann, J; Turecek, P L; Putz, M

    2016-01-01

    BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A. © 2015 Baxalta Innovations GmbH. Haemophilia Published by John Wiley & Sons Ltd.

  10. Efficacy of polyethylene glycol-conjugated bovine granulocyte colony-stimulating factor for reducing the incidence of naturally occurring clinical mastitis in periparturient dairy cows and heifers.

    Science.gov (United States)

    Hassfurther, Renee L; TerHune, Terry N; Canning, Peter C

    2015-03-01

    To evaluate effects of various doses of polyethylene glycol (PEG)-conjugated bovine granulocyte colony-stimulating factor (bG-CSF) on the incidence of naturally occurring clinical mastitis in periparturient dairy cattle. 211 periparturient Holstein cows and heifers. Approximately 7 days before the anticipated date of parturition (day of parturition = day 0), healthy cattle received SC injections of sterile saline (0.9% NaCl) solution (control treatment) or PEG-bG-CSF at 5, 10, or 20 μg/kg. Cattle were commingled and housed in a pen with dirt flooring, which was kept wet to maximize the incidence of naturally occurring clinical mastitis. Within 24 hours after parturition, each animal again received the assigned treatment. Mammary glands and milk were visually scored for abnormalities twice daily for 28 days after parturition. Milk samples were aseptically collected from mammary glands with an abnormal appearance or abnormal milk and submitted for microbial culture. Daily milk production was recorded, and milk composition was assessed on days 3, 5, 7, and 10. Cattle treated with PEG-bG-CSF at 10 and 20 μg/kg had significantly fewer cases of clinical mastitis (9/54 and 5/53, respectively), compared with control cattle (18/53). Administration of PEG -bG-CSF did not significantly affect daily milk production or milk composition. Results suggested that PEG-bG-CSF was effective for reducing the incidence of naturally occurring clinical mastitis in periparturient dairy cattle. Further investigations of the use of PEG-bG-CSF as a potential preventative intervention should be conducted.

  11. Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Therkildsen, M H; Poulsen, Steen Seier; Bretlau, P

    1993-01-01

    Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected...... previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases......., the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus...

  12. Estrogen receptor alpha and nuclear factor Y coordinately regulate the transcription of the SUMO-conjugating UBC9 gene in MCF-7 breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Shibo Ying

    Full Text Available UBC9 encodes a protein that conjugates small ubiquitin-related modifier (SUMO to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the UBC9 gene, we identified and characterized its promoter and cis-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP, and their functional role was confirmed by siRNA knockdown. UBC9 mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of UBC9 mRNA and protein was found in MCF-7 breast cancer cells treated with 17β-estradiol (E2. Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor α (ER-α (one imperfect estrogen response element, ERE and/or nuclear factor Y (NF-Y binding sites (two CCAAT boxes markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E2 treatment and overexpression of ER-α or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the UBC9 promoter in vivo. Furthermore, UBC9 expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal UBC9 promoter and provided evidence that ER-α and NF-Y regulate UBC9 expression on the transcriptional level in response to E2 in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER

  13. Estrogen receptor alpha and nuclear factor Y coordinately regulate the transcription of the SUMO-conjugating UBC9 gene in MCF-7 breast cancer cells.

    Science.gov (United States)

    Ying, Shibo; Dünnebier, Thomas; Si, Jing; Hamann, Ute

    2013-01-01

    UBC9 encodes a protein that conjugates small ubiquitin-related modifier (SUMO) to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the UBC9 gene, we identified and characterized its promoter and cis-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP), and their functional role was confirmed by siRNA knockdown. UBC9 mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of UBC9 mRNA and protein was found in MCF-7 breast cancer cells treated with 17β-estradiol (E2). Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor α (ER-α) (one imperfect estrogen response element, ERE) and/or nuclear factor Y (NF-Y) binding sites (two CCAAT boxes) markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E2 treatment and overexpression of ER-α or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the UBC9 promoter in vivo. Furthermore, UBC9 expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal UBC9 promoter and provided evidence that ER-α and NF-Y regulate UBC9 expression on the transcriptional level in response to E2 in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER

  14. Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis.

    Science.gov (United States)

    Pino, María Teresa Luján; Marotte, Clarisa; Verstraeten, Sandra Viviana

    2017-03-01

    We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF - cells) or the presence (EGF + cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 μM) decreased cell viability in EGF - but not in EGF + cells. In EGF - cells, Tl(I) decreased mitochondrial potential, enhanced H 2 O 2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF - cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF - and EGF + cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF - cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF - and EGF + cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

  15. Epidermal growth factor improves survival and prevents intestinal injury in a murine model of pseudomonas aeruginosa pneumonia.

    Science.gov (United States)

    Dominguez, Jessica A; Vithayathil, Paul J; Khailova, Ludmila; Lawrance, Christopher P; Samocha, Alexandr J; Jung, Enjae; Leathersich, Ann M; Dunne, W Michael; Coopersmith, Craig M

    2011-10-01

    Mortality from pneumonia is mediated, in part, through extrapulmonary causes. Epidermal growth factor (EGF) has broad cytoprotective effects, including potent restorative properties in the injured intestine. The purpose of this study was to determine the efficacy of EGF treatment following Pseudomonas aeruginosa pneumonia. FVB/N mice underwent intratracheal injection of either P. aeruginosa or saline and were then randomized to receive either systemic EGF or vehicle beginning immediately or 24 h after the onset of pneumonia. Systemic EGF decreased 7-day mortality from 65% to 10% when initiated immediately after the onset of pneumonia and to 27% when initiated 24 h after the onset of pneumonia. Even though injury in pneumonia is initiated in the lungs, the survival advantage conferred by EGF was not associated with improvements in pulmonary pathology. In contrast, EGF prevented intestinal injury by reversing pneumonia-induced increases in intestinal epithelial apoptosis and decreases in intestinal proliferation and villus length. Systemic cytokines and kidney and liver function were unaffected by EGF therapy, although EGF decreased pneumonia-induced splenocyte apoptosis. To determine whether the intestine was sufficient to account for extrapulmonary effects induced by EGF, a separate set of experiments was done using transgenic mice with enterocyte-specific overexpression of EGF (IFABP-EGF [intestinal fatty acid-binding protein linked to mouse EGF] mice), which were compared with wild-type mice subjected to pneumonia. IFABP-EGF mice had improved survival compared with wild-type mice following pneumonia (50% vs. 28%, respectively, P < 0.05) and were protected from pneumonia-induced intestinal injury. Thus, EGF may be a potential adjunctive therapy for pneumonia, mediated in part by its effects on the intestine.

  16. Homologous radioimmunoassay for human epidermal growth factor (urogastrone)

    International Nuclear Information System (INIS)

    Dailey, G.E.; Kraus, J.W.; Orth, D.N.

    1978-01-01

    Epidermal growth factor (EGF), a polypeptide hormone originally discovered in the mouse submaxillary gland, stimulates growth in a variety of tissues in several species. This hormone has recently been identified in human urine. A homologous RIA for human EGF (RIA-hEGF) has been developed. In general, levels were similar to those recently reported using a heterologous RIA system. Twenty-four-hour urinary excretion of RIA-hEGF by normal adult males and females was 63.0 +- 3.0 and 52.0 +- 3.5 (mean +- SE) μg/total vol, or 29.7 +- 1.1 and 39.8 +- 1.7 μg/g creatinine, respectively. Excretion by females taking oral contraceptives was significantly greater (60.1 +- 2.7 μg/g creatinine; P 0.05). Several of those with very low values had histories of alcohol abuse. Excretion by patients with Cushing's syndrome was normal. Patients with psoriasis or recovering from major burns excreted both abnormally high and abnormally low levels of RIA-hEGF, with no obvious correlation to their clinical condition. There was no apparent diurnal or postprandial variation in urinary RIA-hEGF excretion by normal subjects. An excellent linear correlation was observed between RIA-hEGF and creatinine concentrations in each urine sample for each subject, suggesting that RIA-hEGF concentration in a random urine sample provides a valid index of 24-h RIA-hEGF excretion

  17. Role of submandibular saliva and epidermal growth factor in gastric cytoprotection

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

    without submandibular glands. Exogenous EGF and saliva with a high but still physiological concentration of EGF significantly reduced the median area in the stomach displaying ulcers and ulcerations, whereas saliva without EGF had no effect. Although EGF is a known inhibitor of gastric acid secretion......The role of submandibular epidermal growth factor in protection of the gastric mucosa was investigated in rats. Removal of the submandibular glands and thereby submandibular epidermal growth factor (EGF) caused rats to develop gastric lesions (ulcerations and ulcers) after administration......, the dose used in the present study had no effect on gastric acid secretion in chronic gastric fistula rats; removal of the submandibular glands also did not have any such effect. We conclude that exocrine secretion of submandibular EGF has a cytoprotective function in the stomach, an effect that may...

  18. Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

    International Nuclear Information System (INIS)

    Hugo, Honor J; Wafai, Razan; Blick, Tony; Thompson, Erik W; Newgreen, Donald F

    2009-01-01

    A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive

  19. Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

    Directory of Open Access Journals (Sweden)

    Thompson Erik W

    2009-07-01

    Full Text Available Abstract Background A feature of epithelial to mesenchymal transition (EMT relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1 and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4 and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4. Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse

  20. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  1. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H 10 . The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  2. Effects of epidermal growth factor on bone formation and resorption in vivo

    International Nuclear Information System (INIS)

    Marie, P.J.; Hott, M.; Perheentupa, J.

    1990-01-01

    The effects of mouse epidermal growth factor (EGF) on bone formation and resorption were examined in male mice. EGF administration (2-200 ng.g-1.day-1 ip for 7 days) induced a dose-dependent rise in plasma EGF levels that remained within physiological range. Histomorphometric analysis of caudal vertebrae showed that EGF (20 and 200 ng.g-1.day-1) reduced the endosteal matrix and mineral appositional rates after 5 days of treatment as measured by double [3H]proline labeling and double tetracycline labeling, respectively. This effect was transitory and was not observed after 7 days of EGF administration. EGF administered for 7 days induced a dose-dependent increase in the periosteal osteoblastic and tetracycline double-labeled surfaces. At high dosage (200 ng.g-1.day-1) EGF administration increased the osteoclastic surface and the number of acid phosphatase-stained osteoclasts, although plasma calcium remained normal. The results show that EGF administration at physiological doses induces distinct effects on endosteal and periosteal bone formation and that the effects are dependent on EGF dosage and duration of treatment. This study indicates that EGF at physiological dosage stimulates periosteal bone formation and increases endosteal bone resorption in the growing mouse

  3. Demonstration of epidermal growth factor binding sites in the adult rat pancreas by light microscopic autoradiography

    International Nuclear Information System (INIS)

    Chabot, J.G.; Walker, P.; Pelletier, G.

    1987-01-01

    The distribution of epidermal growth factor (EGF) receptors was studied in the pancreas using light microscopic autoradiography, which was performed at different time intervals (2-60 min) after injecting 125 I-labeled EGF intravenously into the adult rat. In the exocrine pancreas, a labeling was found to occur over the pyramidal cells of the acini and cells lining the intercalated ducts. Moreover, substantial binding of EGF to cells of the islets of Langerhans was also revealed. At the 2-min time interval, most silver grains were found at the periphery of the target cells. The localization, as well as the diminution of silver grains over the cytoplasm of these cells, between 7 and 60 min, suggested the internalization and degradation of 125 I-labeled EGF. Control experiments indicated that the autoradiography reaction was due to specific interaction of 125 I-labeled EGF with its receptor. These results clearly indicate that EGF receptors are present in the acinar cells and the cells of intercalated ducts of the exocrine pancreas, as well as the cells of the endocrine pancreas. Finding that there are EGF binding sites in pancreatic acinar cells supports the physiological role of EGF in the regulation of pancreatic exocrine function. The presence of EGF receptors in cells of the islets of Langerhans suggests that EGF may play a role in the regulation of the endocrine pancreas

  4. Cellular senescence of human mammary epithelial cells (HMEC) is associated with an altered MMP-7/HB-EGF signaling and increased formation of elastin-like structures.

    Science.gov (United States)

    Bertram, Catharina; Hass, Ralf

    2009-10-01

    The extracellular matrix (ECM) and a complex interplay of cell-to-cell and cell-to-matrix (ECM) interactions provide important platforms to determine cellular senescence and a potentially tumorigenic transformation of normal human mammary epithelial cells (HMEC). An enhanced formation of extracellular filaments, consisting of elastin-like structures, in senescent post-selection HMEC populations was paralleled by a significantly increased expression of its precursor protein tropoelastin and matched with a markedly elevated activity of the cross-linking enzyme family of lysyl oxidases (LOX). RNAi experiments revealed both the ECM metalloproteinase MMP-7 and the growth factor HB-EGF as potential effectors of an increased tropoelastin expression. Moreover, co-localization of MMP-7 and HB-EGF as well as a concomittant downstream signaling via Fra-1 indicated a possible association between the reduced MMP-7 enzyme activity and an impaired HB-EGF processing, resulting in an enhanced tropoelastin synthesis during senescence of HMEC. In agreement with previous work, these findings suggested an important influence of the extracellular proteinase MMP-7 on the aging process of HMEC, affecting both extracellular remodeling as well as intracellular signaling pathways.

  5. Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin

    International Nuclear Information System (INIS)

    Ponec, M.; Kempenaar, J.; Weerheim, A.; Boonstra, J.

    1987-01-01

    We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14 C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression

  6. Gene expression response of A253 human salivary cell line to radiation, Cis-Pt, and EGF

    International Nuclear Information System (INIS)

    Woloschak, G.; Paunesku, T.; Mittal, B.; Dyck, P.; Pauloski, B.; Rademaker, A.; Logemann, J.; Quigg, R.

    2003-01-01

    We are interested in long and short term effects of head and neck cancers treatment, and prior to the studies of patient samples, experiments were designed to observe treatment effects in cultured cells, and examine gene expression profiles from A253 human salivary cells (derived from a head and neck tumor) following exposure to gamma-rays, cisplatin (cis-Pt), and a combination of either with epidermal growth factor (EGF) treatment. A253 cells were treated by: 2 Gy or 10 Gy of γ-rays (Cs137 source, 77 cGy/min), Cis-Pt at 50 μ/mL, and EGF at 40 ng/mL. RNAs were processed and hybridized with Affymetrix Hu95A arrays according to the manufacturer's instructions. Data were scanned and analyzed and we found significant differences in the expression patterns of numerous genes were observed. Some of the more interesting genes are: Requeim [a protein required for apoptosis]; Cyclin D1 (prad1/bcl1) [a cyclin that can function as an oncogene]; FK506 Binding Protein [which may be competing with TGF-beta type I receptor for binding with FK506 thus acting against this powerful immunosuppressant]; Thioredoxin (TXN) [an oxidoreductase with multiple in vitro substrates, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin]; Glutathione Peroxidase (GPX) [whose role in protection against oxidative stress was long ago well documented]; Aquaporin 3 (AQP3) [protein with a water-channel function that was confirmed by functional expression in Xenopus oocytes]; Eukaryotic Initiation Factor 1A (EIF1A) [a translation factor, proposed as a candidate gene for Turner syndrome]; and finally Insulin-like Growth Factor-Binding Protein 6 (IGFBP6) [an autocrine growth inhibitor shown to inhibit growth of HaCat cells and other keratinocyte cell lines

  7. Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

    Science.gov (United States)

    Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

    2008-01-01

    Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B

  8. Optical phase conjugation

    CERN Document Server

    Fisher, Robert A

    1983-01-01

    This book appears at a time of intense activity in optical phase conjugation. We chose not to await the maturation of the field, but instead to provide this material in time to be useful in its development. We have tried very hard to elucidate and interrelate the various nonlinear phenomena which can be used for optical phase conjugation.

  9. Growth of intestinal epithelium in organ culture is dependent on EGF signalling

    International Nuclear Information System (INIS)

    Abud, Helen E.; Watson, Nadine; Heath, Joan K.

    2005-01-01

    Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development

  10. Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

    Directory of Open Access Journals (Sweden)

    Zhong Yao

    Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

  11. The Level of Autoantibodies Targeting Eukaryote Translation Elongation Factor 1 α1 and Ubiquitin-Conjugating Enzyme 2L3 in Nondiabetic Young Adults

    Directory of Open Access Journals (Sweden)

    Eunhee G. Kim

    2016-01-01

    Full Text Available BackgroundThe prevalence of novel type 1 diabetes mellitus (T1DM antibodies targeting eukaryote translation elongation factor 1 alpha 1 autoantibody (EEF1A1-AAb and ubiquitin-conjugating enzyme 2L3 autoantibody (UBE2L3-AAb has been shown to be negatively correlated with age in T1DM subjects. Therefore, we aimed to investigate whether age affects the levels of these two antibodies in nondiabetic subjects.MethodsEEF1A1-AAb and UBE2L3-AAb levels in nondiabetic control subjects (n=150 and T1DM subjects (n=101 in various ranges of age (18 to 69 years were measured using an enzyme-linked immunosorbent assay. The cutoff point for the presence of each autoantibody was determined based on control subjects using the formula: [mean absorbance+3×standard deviation].ResultsIn nondiabetic subjects, there were no significant correlations between age and EEF1A1-AAb and UBE2L3-AAb levels. However, there was wide variation in EEF1A1-AAb and UBE2L3-AAb levels among control subjects <40 years old; the prevalence of both EEF1A1-AAb and UBE2L3-AAb in these subjects was 4.4%. When using cutoff points determined from the control subjects <40 years old, the prevalence of both autoantibodies in T1DM subjects was decreased (EEFA1-AAb, 15.8% to 8.9%; UBE2L3-AAb, 10.9% to 7.9% when compared to the prevalence using the cutoff derived from the totals for control subjects.ConclusionThere was no association between age and EEF1A1-AAb or UBE2L3-AAb levels in nondiabetic subjects. However, the wide variation in EEF1A1-AAb and UBE2L3-AAb levels apparent among the control subjects <40 years old should be taken into consideration when determining the cutoff reference range for the diagnosis of T1DM.

  12. Recycling of epidermal growth factor in a human pancreatic carcinoma cell line

    International Nuclear Information System (INIS)

    Korc, M.; Magun, B.E.

    1985-01-01

    PANC-1 human pancreatic carcinoma cells readily bound and internalized 125 I-labeled epidermal growth factor (EGF). Bound 125 I-labeled EGF was then partially processed to a number of high molecular weight acidic species. Percoll gradient centrifugation of cell homogenates indicated that the majority of 125 I activity localized to several intracellular vesicular compartments. Both intact EGF and its processed species were subsequently released into the incubation medium. A major portion of the released radioactivity was capable of rebinding to the cell. Only a small amount of bound 125 I-labeled EGF was degraded to low molecular weight products, and this degradation was completely blocked by methylamine. These findings suggest that in PANC-1 cells, bound EGF undergoes only limited processing. Both intact EGF and its major processed species bypass the cellular degradative pathways, are slowly released from the cell, and then rebind to the cell

  13. Enhancement of intestinal growth in neonatal rats by epidermal growth factor in milk

    International Nuclear Information System (INIS)

    Berseth, C.L.

    1987-01-01

    Breast milk has been shown to enhance neonatal intestinal growth. Because epidermal growth factor (EGF) is present in the milk of various mammalian species, the hypothesis was tested that EGF in rodent milk mediates, in part, the breast milk-enhanced intestinal growth in neonatal rat. Fifty-eight rat pups fed artificial formal that contained 1.2, 3.0, and 6.0 μg/ml EGF for 39 h had greater incorporation of [ 3 H]thymidine into DNA and DNA content of intestine than 29 pups fed unsupplemented formula. Pups fed EGF for 5 days had significantly greater body weight, intestinal weight, length, and DNA content than control pups. Conversely, pups fed pooled rat milk containing rabbit-derived antibody to EGF for 39 h had intestines of lower weight that contained less DNA than animals fed rat milk containing normal rabbit serum. EGF appears to mediate, in part, breast milk-enhanced neonatal intestinal growth

  14. Efficacy of recombinant bovine epidermal growth factor in the treatment of experimental subclinical Staphylococcus aureus mastitis in a ewe model.

    Science.gov (United States)

    Gabadage, Kamal; Chirino-Trejo, Manuel; Campbell, John; Luby, Christopher

    2017-01-01

    Staphylococcus aureus is the most common contagious mastitis pathogen of dairy cattle. Antimicrobial treatment of infected cattle results in variable cure rates. Epidermal growth factor (EGF) plays an important role in the modulation of host innate immune responses and the regulation of mammary epithelial regeneration, indicating that EGF may be useful as a treatment for mastitis. A pilot study was conducted to evaluate the efficacy of recombinant bovine EGF (rbEGF) for the treatment of S aureus intramammary infection (IMI) using an ovine model. Each ewe was experimentally infected with S aureus in both udder halves. One udder half of each ewe received one of two treatments: EGF (n=13) or pirlimycin (n=13). The contralateral udder half of each ewe received sterile saline as a control. The bacteriological cure rate following rbEGF was significantly lower (15 per cent) than that attained with pirlimycin hydrochloride (61 per cent) and did not differ from that following treatment with sterile saline. Cure rates following treatment with rbEGF were not significantly different to those following sterile saline. Given that EGF is associated with modulation of host immunity and wound healing, future studies into EGF should not focus on whether EGF increases cure rates of S aureus IMI.

  15. Analysis of the principal factors that intervene in the quantification of planar images of uniform distributions of 99mTc by the conjugate view method with background correction by simple subtraction

    International Nuclear Information System (INIS)

    Mora Araya, Luis Diego

    2013-01-01

    The activity of uniform distributions of 99m Tc was quantified by the conjugate view method. The necessary factors of calibration and transmission were calculated to realize the quantification. The dependence of the estimated number of accounts within the source region and variability of the value of the transmission factor were determined, according to the size established for the region of interest, keeping constant its geometry. The images of all acquisitions were corrected by environmental background radiation and radiation dispersed, by the dual energy window method (DEW). The impact of corrections in the image were checked, both qualitatively and quantitative. The acquisition to obtain the calibration factor was realized with the same configuration and the same conditions that were used to realize the acquisition for quantification; in which, the same volume and the same geometry were used to contain the distribution of the activity of 99m Tc. The volume and geometry of the same medium attenuator have obtained a calibration factor exactly in the same circumstances in which have quantified. The behavior of the estimation of the calibration factor of the gamma camera was analyzed, according to the decay corrections of the activity and the attenuation that are applied. The dependence of the calibration factors and transmission were analyzed, according to the region of interest used in the corresponding images to estimate their values. The behavior of the estimation of the activity was determined, according to all possible combinations of the factors studied that have intervened in the quantification algorithm of conjugate view, namely, the size of the region of interest corresponding to the source region, the transmission factor , the calibration factor and background correction by simple subtraction. The results obtained of the estimates of the activity were compared. A tendency is established, indicating which have been combinations of the studied factors that

  16. Is epidermal growth factor involved in development of duodenal polyps in familial polyposis coli?

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1988-01-01

    Duodenal adenomas are a frequent extracolonic manifestation in patients with familial polyposis coli (FPC). Epidermal growth factor (EGF), a polypeptide that stimulates cellular growth and differentiation, is localized in Paneth cells in the small intestine. In two patients with FPC, we found EGF...... immunoreactivity in duodenal adenomas. Numerous EGF immunoreactive Paneth cells were localized, not as usually, in the bottom of the crypts, but scattered along the crypts alone or in clusters. We do not know whether EGF is involved in the development of duodenal polyps in FPC patients, or whether the present...

  17. Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin.

    Directory of Open Access Journals (Sweden)

    Imane Song

    Full Text Available One week of treatment with EGF and gastrin (EGF/G was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of

  18. In vivo tumor targeting and imaging with anti-vascular endothelial growth factor antibody-conjugated dextran-coated iron oxide nanoparticles

    Directory of Open Access Journals (Sweden)

    Hsieh WJ

    2012-06-01

    Full Text Available Wan-Ju Hsieh,1 Chan-Jung Liang,1 Jen-Jie Chieh,4 Shu-Huei Wang,1 I-Rue Lai,1 Jyh-Horng Chen,2 Fu-Hsiung Chang,3 Wei-Kung Tseng,4–6 Shieh-Yueh Yang,4 Chau-Chung Wu,7 Yuh-Lien Chen11Institute of Anatomy and Cell Biology, College of Medicine, 2Department of Electrical Engineering, 3Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan; 4Institute of Electro-Optical Science and Technology, National Taiwan Normal University, Taipei, Taiwan; 5Division of Cardiology, Department of Internal Medicine, E-Da Hospital, Taipei, Taiwan; 6Department of Medical Imaging and Radiological Sciences, I-Shou University, Taipei, Taiwan; 7Department of Internal Medicine and Primary Care Medicine, National Taiwan University Hospital, Taipei, TaiwanBackground: Active targeting by specific antibodies combined with nanoparticles is a promising technology for cancer imaging and detection by magnetic resonance imaging (MRI. The aim of the present study is to investigate whether the systemic delivery of antivascular endothelial growth factor antibodies conjugating to the surface of functionalized supermagnetic iron oxide nanoparticles (anti-VEGF-NPs led to target-specific accumulation in the tumor.Methods: The VEGF expression in human colon cancer and in Balb/c mice bearing colon cancers was examined by immunohistochemistry. The distribution of these anti-VEGF-NPs particles or NPs particles were evaluated by MRI at days 1, 2, or 9 after the injection into the jugular vein of Balb/c mice bearing colon cancers. Tumor and normal tissues (liver, spleen, lung, and kidney were collected and were examined by Prussian blue staining to determine the presence and distribution of NPs in the tissue sections.Results: VEGF is highly expressed in human and mouse colon cancer tissues. MRI showed significant changes in the T*2 signal and T2 relaxation in the anti-VEGF-NP- injected-mice, but not in mice injected with NP alone. Examination of paraffin

  19. Qualidade conjugal: mapeando conceitos

    Directory of Open Access Journals (Sweden)

    Clarisse Mosmann

    2006-12-01

    Full Text Available Apesar da ampla utilização do conceito de qualidade conjugal, identifica-se falta de clareza conceitual acerca das variáveis que o compõem. Esse artigo apresenta revisão da literatura na área com o objetivo de mapear o conceito de qualidade conjugal. Foram analisadas sete principais teorias sobre o tema: Troca Social, Comportamental, Apego, Teoria da Crise, Interacionismo Simbólico. Pelos postulados propostos nas diferentes teorias, podem-se identificar três grupos de variáveis fundamentais na definição da qualidade conjugal: recursos pessoais dos cônjuges, contexto de inserção do casal e processos adaptativos. Neste sentido, a qualidade conjugal é resultado do processo dinâmico e interativo do casal, razão deste caráter multidimensional.

  20. Conjugate Gaze Palsies

    Science.gov (United States)

    ... version Home Brain, Spinal Cord, and Nerve Disorders Cranial Nerve Disorders Conjugate Gaze Palsies Horizontal gaze palsy Vertical ... Version. DOCTORS: Click here for the Professional Version Cranial Nerve Disorders Overview of the Cranial Nerves Internuclear Ophthalmoplegia ...

  1. Conjugated Polymer Solar Cells

    National Research Council Canada - National Science Library

    Paraschuk, Dmitry Y

    2006-01-01

    This report results from a contract tasking Moscow State University as follows: Conjugated polymers are promising materials for many photonics applications, in particular, for photovoltaic and solar cell devices...

  2. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  3. Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

    Science.gov (United States)

    Zhang, Xu; Belkina, Natalya; Jacob, Harrys Kishore Charles; Maity, Tapan; Biswas, Romi; Venugopalan, Abhilash; Shaw, Patrick G; Kim, Min-Sik; Chaerkady, Raghothama; Pandey, Akhilesh; Guha, Udayan

    2015-01-01

    Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http

  4. Modulation of Regorafenib effects on HCC cell lines by epidermal growth factor.

    Science.gov (United States)

    D'Alessandro, Rosalba; Refolo, Maria Grazia; Lippolis, Catia; Carella, Nicola; Messa, Caterina; Cavallini, Aldo; Carr, Brian Irving

    2015-06-01

    Blood platelet numbers are correlated to growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) also stimulated growth and migration, and antagonized the growth-inhibitory and apoptotic effects of both Sorafenib and Regorafenib, two multikinase inhibitors, on three HCC cell lines. In this study, in vitro function of human epidermal growth factor (EGF) with and without Sorafenib or Regorafenib was investigated. An ELISA kit was used to evaluate the EGF concentrations in hPLs. In vitro function of EGF was assessed with proliferation MTT test. Apoptosis assay, scratch assays, and Transwell assays were performed for apoptosis, invasion, and migration, respectively. MAPK Activation Kit was used to explore MAPK phosphorylation. EGF antagonized the growth inhibition of Regorafenib on three HCC cell lines. Regorafenib-mediated growth inhibition was blocked by 70 % when the cells were pre-treated with EGF. EGF also blocked Regorafenib-induced apoptosis, as well as Regorafenib-induced decreases in cell migration and invasion. The EGF effects were in turn antagonized by concomitant addition to the cultures of EGF receptor antagonist Erlotinib, showing that the EGF receptor was involved in the mechanisms of EGF-mediated blocking of Regorafenib effects. Erlotinib also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that EGF was an important component of hPL actions. All these results show that EGF antagonized Regorafenib-mediated growth and migration inhibition and apoptosis induction in HCC cells and reinforce the idea that microenvironment can influence cancer drug actions.

  5. Morphology and dynamics of tumor cell colonies propagating in epidermal growth factor supplemented media

    Science.gov (United States)

    Muzzio, N. E.; Carballido, M.; Pasquale, M. A.; González, P. H.; Azzaroni, O.; Arvia, A. J.

    2018-07-01

    The epidermal growth factor (EGF) plays a key role in physiological and pathological processes. This work reports on the influence of EGF concentration (c EGF) on the modulation of individual cell phenotype and cell colony kinetics with the aim of perturbing the colony front roughness fluctuations. For this purpose, HeLa cell colonies that remain confluent along the whole expansion process with initial quasi-radial geometry and different initial cell populations, as well as colonies with initial quasi-linear geometry and large cell population, are employed. Cell size and morphology as well as its adhesive characteristics depend on c EGF. Quasi-radial colonies (QRC) expansion kinetics in EGF-containing medium exhibits a complex behavior. Namely, at the first stages of growth, the average QRC radius evolution can be described by a t 1/2 diffusion term coupled with exponential growth kinetics up to a critical time, and afterwards a growth regime approaching constant velocity. The extension of each regime depends on c EGF and colony history. In the presence of EGF, the initial expansion of quasi-linear colonies (QLCs) also exhibits morphological changes at both the cell and the colony levels. In these cases, the cell density at the colony border region becomes smaller than in the absence of EGF and consequently, the extension of the effective rim where cell duplication and motility contribute to the colony expansion increases. QLC front displacement velocity increases with c EGF up to a maximum value in the 2–10 ng ml‑1 range. Individual cell velocity is increased by EGF, and an enhancement in both the persistence and the ballistic characteristics of cell trajectories can be distinguished. For an intermediate c EGF, collective cell displacements contribute to the roughening of the colony contours. This global dynamics becomes compatible with the standard Kardar–Parisi–Zhang growth model, although a faster colony roughness saturation in EGF-containing medium

  6. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Energy Technology Data Exchange (ETDEWEB)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Rodrigues, Michele A. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Department of General Pathology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Gomes, Dawidson A., E-mail: dawidson@ufmg.br [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil)

    2016-09-09

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  7. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    International Nuclear Information System (INIS)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.; Rodrigues, Michele A.; Gomes, Dawidson A.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  8. Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis.

    Science.gov (United States)

    Clark, Jessica A; Gan, Heng; Samocha, Alexandr J; Fox, Amy C; Buchman, Timothy G; Coopersmith, Craig M

    2009-09-01

    Systemic administration of epidermal growth factor (EGF) decreases mortality in a murine model of septic peritonitis. Although EGF can have direct healing effects on the intestinal mucosa, it is unknown whether the benefits of systemic EGF in peritonitis are mediated through the intestine. Here, we demonstrate that enterocyte-specific overexpression of EGF is sufficient to prevent intestinal barrier dysfunction and improve survival in peritonitis. Transgenic FVB/N mice that overexpress EGF exclusively in enterocytes (IFABP-EGF) and wild-type (WT) mice were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability, expression of the tight junction proteins claudins-1, -2, -3, -4, -5, -7, and -8, occludin, and zonula occludens-1; villus length; intestinal epithelial proliferation; and epithelial apoptosis were evaluated. A separate cohort of mice was followed for survival. Peritonitis induced a threefold increase in intestinal permeability in WT mice. This was associated with increased claudin-2 expression and a change in subcellular localization. Permeability decreased to basal levels in IFABP-EGF septic mice, and claudin-2 expression and localization were similar to those of sham animals. Claudin-4 expression was decreased following CLP but was not different between WT septic mice and IFABP-EGF septic mice. Peritonitis-induced decreases in villus length and proliferation and increases in apoptosis seen in WT septic mice did not occur in IFABP-EGF septic mice. IFABP-EGF mice had improved 7-day mortality compared with WT septic mice (6% vs. 64%). Since enterocyte-specific overexpression of EGF is sufficient to prevent peritonitis-induced intestinal barrier dysfunction and confers a survival advantage, the protective effects of systemic EGF in septic peritonitis appear to be mediated in an intestine-specific fashion.

  9. Epidermal Growth Factor Improves Intestinal Integrity and Survival in Murine Sepsis Following Chronic Alcohol Ingestion.

    Science.gov (United States)

    Klingensmith, Nathan J; Yoseph, Benyam P; Liang, Zhe; Lyons, John D; Burd, Eileen M; Margoles, Lindsay M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

    2017-02-01

    Epidermal growth factor (EGF) is a cytoprotective protein that improves survival in preclinical models of sepsis through its beneficial effects on intestinal integrity. Alcohol use disorder worsens intestinal integrity and is associated with increased morbidity and mortality in critical illness. We sought to determine whether chronic alcohol ingestion alters the host response to systemic administration of EGF in sepsis. Six-week-old FVB/N mice were randomized to receive 20% alcohol or water for 12 weeks. All mice then underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive either intraperitoneal injection of EGF (150 μg/kg/day) or normal saline. Water-fed mice given EGF had decreased 7-day mortality compared with water-fed mice (18% vs. 55%). Alcohol-fed mice given EGF also had decreased 7-day mortality compared with alcohol-fed mice (48% vs. 79%). Notably, while systemic EGF improved absolute survival to a similar degree in both water-fed and alcohol-fed mice, mortality was significantly higher in alcohol+EGF mice compared with water+EGF mice. Compared with water-fed septic mice, alcohol-fed septic mice had worsened intestinal integrity with intestinal hyperpermeability, increased intestinal epithelial apoptosis, decreased proliferation and shorter villus length. Systemic administration of EGF to septic alcohol-fed mice decreased intestinal permeability compared with septic alcohol-fed mice given vehicle, with increased levels of the tight junction mediators claudin-5 and JAM-A. Systemic administration of EGF to septic alcohol-fed mice also decreased intestinal apoptosis with an improvement in the Bax/Bcl-2 ratio. EGF also improved both crypt proliferation and villus length in septic alcohol-fed mice. EGF administration resulted in lower levels of both pro- and anti-inflammatory cytokines monocyte chemoattractant protein-1, tumor necrosis factor, and interleukin 10 in alcohol-fed mice. EGF is therefore

  10. Possible physiological role of milk epidermal growth factor in neonatal eyelid opening

    International Nuclear Information System (INIS)

    Tsutsumi, O.; Tsutsumi, A.; Oka, T.

    1987-01-01

    The eyelid opening of newborn mice occurs normally on day 13.9 +/- 1.8 after birth. When newborn mice were injected with anti-epidermal growth factor (EGF) antibody every other day starting on day 1 after birth, the eyelid opening was delayed by ∼ 3 days. The effect of anti-EGF became less prominent as the treatment was started at later times: when it was give from day 7, no delay in eyelid opening was observed. On the other hand, eyelid opening was enhanced by ∼ 3 days by EGF injection given on day 3 for every other day. This effect of EGF was antagonized by simultaneous administration of anti-EGF antibody. EGF was present at a concentration of 6.6 ng/ml in the plasma of 1-wk-old pups nursed by their mother, but it was not detectable in the plasma of 3-wk-old weaned pups. EGF concentration in the submandibular glands, however, was 17 times greater in 3- than in 1-wk-old pups. EGF was measured by radioimmunoassay. These results suggest that milk EGF may play a physiological role in eyelid opening during the neonatal period

  11. Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

    International Nuclear Information System (INIS)

    Pickhard, Anja C; Schlegel, Jürgen; Arnold, Wolfgang; Reiter, Rudolf; Margraf, Johanna; Knopf, Andreas; Stark, Thomas; Piontek, Guido; Beck, Carolin; Boulesteix, Anne-Laure; Scherer, Elias Q; Pigorsch, Steffi

    2011-01-01

    Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy

  12. Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling networks

    DEFF Research Database (Denmark)

    Hammond, Dean E; Hyde, Russell; Kratchmarova, Irina

    2010-01-01

    549 lung adenocarcinoma cells with EGF or HGF. In total, we obtained quantitative information for 274 proteins, which respond to either or both stimuli by >1.5 fold changes in enrichment, following immuno-precipitation with antiphosphotyrosine antibodies. The data reveal a high degree of overlap...

  13. Expression of polyhedrin-hEGF fusion protein in cultured cells and ...

    African Journals Online (AJOL)

    GRACE

    2006-06-02

    Jun 2, 2006 ... EGF virus and wild virus protected the gastric mucosa against ethanol-induced damage in rats, although .... virus stock was prepared and the dilution of the virus was calculated .... silkworm larvae presented typical symptoms of BmNPV .... Plasmodium falciparum circumsporozoite protein malaria vaccine.

  14. Near Infrared Optical Visualization of Epidermal Growth Factor Receptors Levels in COLO205 Colorectal Cell Line, Orthotopic Tumor in Mice and Human Biopsies

    Directory of Open Access Journals (Sweden)

    Philip Lazarovici

    2013-07-01

    Full Text Available In this study, we present the applicability of imaging epidermal growth factor (EGF receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR. The near infrared (NIR bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

  15. LPA, HGF, and EGF utilize distinct combinations of signaling pathways to promote migration and invasion of MDA-MB-231 breast carcinoma cells

    International Nuclear Information System (INIS)

    Harrison, Susan MW; Knifley, Teresa; Chen, Min; O’Connor, Kathleen L

    2013-01-01

    Various pathways impinge on the actin-myosin pathway to facilitate cell migration and invasion including members of the Rho family of small GTPases and MAPK. However, the signaling components that are considered important for these processes vary substantially within the literature with certain pathways being favored. These distinctions in signaling pathways utilized are often attributed to differences in cell type or physiological conditions; however, these attributes have not been systematically assessed. To address this question, we analyzed the migration and invasion of MDA-MB-231 breast carcinoma cell line in response to various stimuli including lysophosphatidic acid (LPA), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) and determined the involvement of select signaling pathways that impact myosin light chain phosphorylation. LPA, a potent stimulator of the Rho-ROCK pathway, surprisingly did not require the Rho-ROCK pathway to stimulate migration but instead utilized Rac and MAPK. In contrast, LPA-stimulated invasion required Rho, Rac, and MAPK. Of these three major pathways, EGF-stimulated MDA-MB-231 migration and invasion required Rho; however, Rac was essential only for invasion and MAPK was dispensable for migration. HGF signaling, interestingly, utilized the same pathways for migration and invasion, requiring Rho but not Rac signaling. Notably, the dependency of HGF-stimulated migration and invasion as well as EGF-stimulated invasion on MAPK was subject to the inhibitors used. As expected, myosin light chain kinase (MLCK), a convergence point for MAPK and Rho family GTPase signaling, was required for all six conditions. These observations suggest that, while multiple signaling pathways contribute to cancer cell motility, not all pathways operate under all conditions. Thus, our study highlights the plasticity of cancer cells to adapt to multiple migratory cues

  16. Role of growth factors in the growth of normal and transformed cells

    International Nuclear Information System (INIS)

    Lokeshwar, V.B.

    1989-01-01

    Growth factors play an important role in the growth of normal cells. However, their untimely and/or excess production leads to neoplastic transformation. The role of growth factors in the growth of normal cells was studied by investigating the mechanism of transmodulation of the cell surface EGF receptor number by protamine. Protamine increased the EGF stimulated mitogenic response in Swiss mouse 3T3 cells and A431 cells by increasing the number of functionally active EGF receptors. Protamine also increased EGF receptor number in plasma membranes and solubilized membranes. This was evidenced by an increase in both 125 I-EGF-EGF-receptor complex and EGF stimulated phosphorylation of the EGF receptor. The solubilized EGF receptor was retained on a protamine-agarose gel indicating that protamine might increase EGF receptor number by directly activating cryptic EGF receptors in the plasma membranes. The role of growth factors in neoplastic transformation was studied by investigating the role of the oncogene v-sis in the growth of Simian sarcoma virus (SSV) transformed cells. The product of the oncogene v-sis is 94% homologous to the B chain of PDGF. This study found that (i) v-sis gene product is synthesized as a 32 kDa unglycosylated monomer which is glycosylated, dimerized and proteolytically processed into p36, p72, p68, p58, p44 and p27 mol. wt. species respectively. (ii) p36, p72, p68 and p58 are very likely formed in the endoplasmic reticulum and/or Golgi complex. A fraction of newly synthesized p72, p68 and p58 is degraded intracellularly at a fast rate. (iii) p44 is a secretory product which remains tightly associated with the cell surface. p44 is recaptured by the cells through interaction with cell surface PDGF receptors and degraded into p27. (iv) During long term cultures p44 is extracellularly cleaved into a 27 kDa product

  17. Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Su, Zhongchun; Ma, Huan; Wu, Zhengzheng [Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632 (China); Zeng, Huilan [Department of Hematology, The First Affiliated Hospital, Jinan University, Guangzhou 510632 (China); Li, Zhizhong [Department of Bone, The First Affiliated Hospital, Jinan University, Guangzhou 510632 (China); Wang, Yuechun; Liu, Gexiu [Department of Physiology, School of Medicine, Jinan University, Guangzhou 510632 (China); Xu, Bin; Lin, Yongliang; Zhang, Peng [Grandhope Biotech Co., Ltd., Building D, #408, Guangzhou International Business Incubator, Guangzhou Science Park, Guangzhou 510663, Guangdong (China); Wei, Xing, E-mail: wei70@hotmail.com [Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632 (China)

    2014-11-01

    Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin–Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing. - Highlights: • HA can increase the adsorption of EGF to decellularized scaffolds. • HA can sustain the release of EGF from

  18. Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

    Science.gov (United States)

    Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

    2011-07-01

    Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

  19. Immunohistochemical localisation and developmental aspects of epidermal growth factor in the rat

    DEFF Research Database (Denmark)

    Raaberg, L; Nexø, E; Damsgaard Mikkelsen, J

    1988-01-01

    The tissue localisation and time of first appearance of Epidermal Growth Factor (EGF) in the developing rat were investigated by means of immunohistochemistry, radioimmunoassay and radioreceptor assay. In this study we were able to show, that EGF appears prenatally in the lung and the kidney from...

  20. Does epidermal growth factor play a role in the action of sucralfate?

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier

    1987-01-01

    Epidermal growth factor (EGF) is a mitogenic peptide synthesized in the submandibular glands and released in saliva. EGF is able to prevent the development of gastrointestinal ulcers in the rat and to accelerate their healing. The present work was undertaken to examine whether Sucralfate acts via...

  1. Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

    1991-01-01

    The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

  2. The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF

    DEFF Research Database (Denmark)

    Zhou, Shuling; Ochalek, Anna; Szczesna, Karolina

    2016-01-01

    Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens, including basic fibroblast growth factor (bFGF) and epidermal growth...... factor (EGF). However, these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here, we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations...... of the neural rosettes, resulting in subsequent cholinergic neuron differentiation. Thus, our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells....

  3. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan, E-mail: jan@metabol.su.se

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR

  4. Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

    Science.gov (United States)

    Phuc, Le Thi Minh; Taniguchi, Akiyoshi

    2017-06-19

    The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

  5. Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1991-01-01

    Epidermal growth factor receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization

  6. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  7. Photoluminescence in conjugated polymers

    International Nuclear Information System (INIS)

    Furst, J.E.; Laugesen, R.; Dastoor, P.; McNeill, C.

    2002-01-01

    Full text: Conjugated polymers combine the electronic and optical properties of semiconductors with the processability of polymers. They contain a sequence of alternate single and double carbon bonds so that the overlap of unhybridised p z orbitals creates a delocalised ρ system which gives semiconducting properties with p-bonding (valence) and p* -antibonding (conduction) bands. Photoluminesence (PL) in conjugated polymers results from the radiative decay of singlet excitons confined to a single chain. The present work is the first in a series of studies in our laboratory that will characterize the optical properties of conjugated polymers. The experiment involves the illumination of thin films of conjugated polymer with UV light (I=360 nm) and observing the subsequent fluorescence using a custom-built, fluorescence spectrometer. Photoluminesence spectra provide basic information about the structure of the polymer film. A typical spectrum is shown in the accompanying figure. The position of the first peak is related to the polymer chain length and resolved multiple vibronic peaks are an indication of film structure and morphology. We will also present results related to the optical degradation of these materials when exposed to air and UV light

  8. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2018-05-15

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  9. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  10. Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

    DEFF Research Database (Denmark)

    Ejskjaer, Kirsten; Sørensen, B S; Poulsen, Steen Seier

    2005-01-01

    The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system....... Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1...... (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show...

  11. EGF-CFC proteins are essential coreceptors for the TGF-β signals Vg1 and GDF1

    Science.gov (United States)

    Cheng, Simon K.; Olale, Felix; Bennett, James T.; Brivanlou, Ali H.; Schier, Alexander F.

    2003-01-01

    The TGF-β signals Nodal, Activin, GDF1, and Vg1 have been implicated in mesoderm induction and left-right patterning. Nodal and Activin both activate Activin receptors, but only Nodal requires EGF-CFC coreceptors for signaling. We report that Vg1 and GDF1 signaling in zebrafish also depends on EGF-CFC proteins, but not on Nodal signals. Correspondingly, we find that in Xenopus Vg1 and GDF1 bind to and signal through Activin receptors only in the presence of EGF-CFC proteins. These results establish that multiple TGF-β signals converge on Activin receptor/EGF-CFC complexes and suggest a more widespread requirement for coreceptors in TGF-β signaling than anticipated previously. PMID:12514096

  12. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    Directory of Open Access Journals (Sweden)

    Mary M Melia

    Full Text Available Signalling lymphocyte activation molecule (SLAM has been identified as an immune cell receptor for the morbilliviruses, measles (MV, canine distemper (CDV, rinderpest and peste des petits ruminants (PPRV viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4, also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF,for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  13. Bio-imaging of colorectal cancer models using near infrared labeled epidermal growth factor.

    Directory of Open Access Journals (Sweden)

    Gadi Cohen

    Full Text Available Novel strategies that target the epidermal growth factor receptor (EGFR have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

  14. Modulation of cultured porcine granulosa cell responsiveness to follicle stimulating hormone and epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Buck, P.A.

    1986-01-01

    Ovarian follicular development is dependent upon the coordinated growth and differentiation of the granulosa cells which line the follicle. Follicle stimulating hormone (FSH) induces granulosa cell differentiation both in vivo and in vitro. Epidermal growth factor (EGF) stimulates granulosa cell proliferation in vitro. The interaction of these two effectors upon selected parameters of growth and differentiation was examined in monolayer cultures of porcine granulose cells. Analysis of the EGF receptor by /sup 125/I-EGF binding revealed that the receptor was of high affinity with an apparent dissociation constant of 4-6 x 10/sup -10/ M. The average number of receptors per cell varied with the state of differentiation both in vivo and in vitro; highly differentiated cells bound two-fold less /sup 125/I-EGF and this effect was at least partially induced by FSH in vitro. EGF receptor function was examined by assessing EGF effects on cell number and /sup 3/H-thymidine incorporation. EGF stimulated thymidine incorporation in both serum-free and serum-supplemented culture, but only in serum-supplemented conditions was cell number increased. EGF receptor function was inversely related to the state of differentiation and was attenuated by FSH. The FSH receptor was examined by /sup 125/I-FSH binding. EGF increased FSH receptor number, and lowered the affinity of the receptor. The function of these receptors was assessed by /sup 125/I-hCG binding and progesterone radioimmunoassay. If EGF was present continuously in the cultures. FSH receptor function was attenuated regardless of FSH receptor number. A preliminary effort to examine the mechanism of this interaction was performed by analyzing hormonally controlled protein synthesis with /sup 35/S-methionine labeling, SDS polyacrylamide gel electrophoresis and fluorography. FSH promoted the expression of a 27,000 dalton protein. This effect was attenuated by EGF.

  15. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  16. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  17. The critical role of EGF-β-catenin signaling in the epithelial-mesenchymal transition in human glioblastoma

    Directory of Open Access Journals (Sweden)

    Wang X

    2017-05-01

    Full Text Available Xingqiang Wang, Shanshi Wang, Xiaolong Li, Shigang Jin, Feng Xiong, Xin Wang Department of Neurosurgery, People’s Hospital of Rizhao, Jining Medical University, Rizhao, China Abstract: To date, β-catenin has been reported to be implicated in mediating the epithelial-mesenchymal transition (EMT in a variety of human cancers, which can be triggered by EGF. However, the mechanisms underlying EGF-β-catenin pathway-induced EMT of glioblastoma multiforme (GBM have not been reported previously. In the present study, immunohistochemistry, reverse transcription polymerase chain reaction, and Western blot were applied to investigate the effect of EGF-β-catenin pathway on EMT of GBM. Here, we identified that β-catenin mRNA and protein levels were up-regulated in GBM tissues and four kinds of glioblastoma cell lines, including T98G, A172, U87, and U251 cells, compared with normal brain tissue and astrocytes. In U87 cell line, inhibition of β-catenin by siRNA suppressed EGF-induced proliferation, migration, invasiveness, and the expression of EMT activators (Snail and Slug. In addition, the expression of epithelial markers (E-cadherin was up-regulated and the expression of mesenchymal markers (N-cadherin and MMP9 was down-regulated. Finally, inhibitor of PI3K/Akt signaling pathways inactivated the EGF-β-catenin-induced EMT. In conclusion, β-catenin-EMT pathway induced by EGF is important for GBM progression by the PI3K/Akt pathways. Inhibition of β-catenin leads to suppression of EGF pathway-induced EMT, which provides a new way to treat GBM patients. Keywords: EGF, β-catenin, EMT, GBM

  18. Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

    DEFF Research Database (Denmark)

    Thulesen, Jesper; Bor, Mustafa Vakur; Thulesen, Stina

    2002-01-01

    The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta...

  19. Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

    1990-01-01

    Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

  20. ErbB1-4-dependent EGF/neuregulin signals and their cross talk in the central nervous system: pathological implications in schizophrenia and Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Yuriko eIwakura

    2013-02-01

    Full Text Available Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinson’s disease. Among the ErbB receptors, ErbB1 and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes and their precursors. Thus, deficits in ErbB signaling might contribute to schizophrenia neuropathology stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines that mimic EGF and neuregulin-1 in brain diseases are also discussed.

  1. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  2. Gene and protein expression of epidermal growth factor measured on the kidney 24 hours after irradiation correlates to late radiation damage

    International Nuclear Information System (INIS)

    Otsuka, Makoto; Hatakenaka, Masamitsu

    2001-01-01

    This study was designed to evaluate the proliferative response of epidermal growth factor (EGF) gene expression as an early indicator of late renal radiation damage. EGF gene expression was measured in the irradiated left kidney of C3H/HeSlc mice using RT-PCR 24 hours after radiation doses of 9, 12, or 15 Gy. In a second experiment, the same radiation doses were administered to the right kidney plus the lower half of the left kidney. The partly irradiated left kidneys were harvested and EGF gene expression was measured. The irradiated whole right kidneys were subjected to immunohistochemical staining for EGF protein. In a third experiment, 12 Gy was administered to the right kidney plus the lower half of the left kidney. The mice underwent left nephrectomy 24 hours after radiation, and the EGF gene expression in the kidney was correlated with the blood urea nitrogen (BUN) level representing late renal functional damage. EGF expression increased in 1 of 10 control mice and in 9 of 10 mice that received 15 Gy. The extent of increase of EGF was dependent on radiation dose. In mice having an increased BUN level after irradiation, 7 of 10 had EGF positive irradiated kidneys. All six mice whose BUN levels were unchanged had EGF-negative irradiated kidneys. EGF protein staining was observed in tubule cells only, not in glomerular cells. The amount of EGF protein staining correlated with radiation dose to some extent. EGF gene expression seems to be a very early indicator of late radiation damage to the kidney. (author)

  3. Four novel FBN1 mutations: Significance for mutant transcript level and EGF-like domain calcium binding in the pathogenesis of Marfan syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Dietz, H.C.; McIntosh, I.; Pyeritz, R.E.; Francomano, C.A. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)); Sakai, L.Y.; Corson, G.M.; Chalberg, S.C. (Oregon Health Sciences Univ., Portland (United States))

    1993-08-01

    Defects of fibrillin (FBN1), a glycoprotein component of the extracellular microfibril, cause Marfan syndrome. This disorder is characterized by marked inter- and intrafamilial variation in phenotypic severity. To understand the molecular basis for this clinical observation, the authors have screened the fibrillin gene (FBN1) on chromosome 15, including the newly cloned 5[prime] coding sequence, for disease-producing alterations in a panel of patients with a wide range of manifestations and clinical severity. All the missense mutations identified to date, including two novel mutations discussed here, are associated with classic and moderate to severe disease and occur at residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. In contrast, two new mutations that create premature signals for termination of translation of mRNA and are associated with reduction in the amount of mutant allele transcript produce a range of phenotypic severity. The patient with the lowest amount of mutant transcript has the mildest disease. These data support a role for altered calcium binding to EGF-like domains in the pathogenesis of Marfan syndrome and suggest a dominant negative mechanism for the pathogenesis of this disorder. 26 refs., 6 figs., 1 tab.

  4. Synergistic Induction of Cyclooxygenase-2 by Transforming Growth Factor-β1 and Epidermal Growth Factor Inhibits Apoptosis in Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Debabrata Saha

    1999-12-01

    Full Text Available Increased expression of cyclooxygenase-2 (COX-2 expression has been observed in several human tumor types and in selected animal and cell culture models of carcinogenesis, including lung cancer. Increased expression of COX-2 and production of prostaglandins appear to provide a survival advantage to transformed cells through the inhibition of apoptosis, increased attachment to extracellular matrix, increased invasiveness, the stimulation of angiogenesis. In the present studies, we found that transforming growth factor β1 (TGF-β1 and epidermal growth factor (EGF synergistically induced the expression of COX-2 and prostaglandin E2 (PGE2 production in mink lung epithelial (Mvi Lu cells. EGF, but not PDGF or IGF-1, was able to inhibit TGF-β1-induced apoptosis in Mvi Lu cells and this effect was blocked by NS-398, a selective inhibitor of COX-2 activity, suggesting a possible role for COX-2 in the anti-apoptosic effect of EGF receptor ligands. The combination of TGF-β1 and EGF also significantly induced COX-2 expression in rat intestinal epithelial (RIE-1 cells and completely prevented sodium butyrate (NaBu-induced apoptosis. The synergistic induction of COX-2 by TGF-β1 and EGF was not observed in R1B-L17 cells, a line derived from Mvi Lu cells that lacks the TGF-β type-I receptor. AG1478, a selective inhibitor of EGF receptor tyrosine kinase activity, completely suppressed the induction of COX-2 expression by either EGF or TGF-β1+EGF. Also, PD98059, a specific inhibitor of MEK/ERK pathway, SB203580, a specific inhibitor of p38 MAPK activity, significantly inhibited the induction of COX-2 in response to combined EGF and TGF-β1. These results suggest an important collaborative interaction of TGF-β1 and EGF signaling in the induction of COX-2 and prostaglandin production in Mv1Lu cells.

  5. Expression of the EGF Family in Gastric Cancer

    DEFF Research Database (Denmark)

    Nielsen, Trine Ostergaard; Friis-Hansen, Lennart; Poulsen, Steen Seier

    2014-01-01

    Gastric cancer is a major cause of cancer-related deaths in both men and women. The epidermal growth factor receptors are EGFR, HER2, HER3 and HER4. Of the four epidermal growth factor receptors, EGFR and HER2 are well-known oncogenes involved in gastric cancer. Little, however, is known about...... the role played by HER3 and HER4 in this disease. We obtained paired samples from the tumor and the adjacent normal tissue from the same patient undergoing surgery for gastric cancer. Using RT-qPCR, we quantified the mRNA expression of the four receptors including the HER4 splicing isoforms and all....... These results support the involvement of EGFR and HER2 in gastric cancer and suggest an interesting association of reduced HER4 expression with development of gastric cancer....

  6. Polymer carriers for anticancer drugs targeted to EGF receptor

    Czech Academy of Sciences Publication Activity Database

    Studenovský, Martin; Pola, Robert; Pechar, Michal; Etrych, Tomáš; Ulbrich, Karel; Kovář, Lubomír; Kabešová, Martina; Říhová, Blanka

    2012-01-01

    Roč. 12, č. 12 (2012), s. 1714-1720 ISSN 1616-5187 R&D Projects: GA AV ČR IAAX00500803 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : biocompatibility * EGFR * peptides Subject RIV: CD - Macromolecular Chemistry; EC - Immunology (MBU-M) Impact factor: 3.742, year: 2012

  7. Double emulsion electrospun nanofibers as a growth factor delivery vehicle for salivary gland regeneration

    Science.gov (United States)

    Foraida, Zahraa I.; Sharikova, Anna; Peerzada, Lubna N.; Khmaladze, Alexander; Larsen, Melinda; Castracane, James

    2017-08-01

    Sustained delivery of growth factors, proteins, drugs and other biologically active molecules is necessary for tissue engineering applications. Electrospun fibers are attractive tissue engineering scaffolds as they partially mimic the topography of the extracellular matrix (ECM). However, they do not provide continuous nourishment to the tissue. In search of a biomimetic scaffold for salivary gland tissue regeneration, we previously developed a blend nanofiber scaffold composed of the protein elastin and the synthetic polymer polylactic-co-glycolic acid (PLGA). The nanofiber scaffold promoted in vivo-like salivary epithelial cell tissue organization and apicobasal polarization. However, in order to enhance the salivary cell proliferation and biomimetic character of the scaffold, sustained growth factor delivery is needed. The composite nanofiber scaffold was optimized to act as a growth factor delivery system using epidermal growth factor (EGF) as a model protein. The nanofiber/EGF hybrid nanofibers were synthesized by double emulsion electrospinning where EGF is emulsified within a water/oil/water (w/o/w) double emulsion system. Successful incorporation of EGF was confirmed using Raman spectroscopy. EGF release profile was characterized using enzyme-linked immunosorbent assay (ELIZA) of the EGF content. Double emulsion electrospinning resulted in slower release of EGF. We demonstrated the potential of the proposed double emulsion electrospun nanofiber scaffold for the delivery of growth factors and/or drugs for tissue engineering and pharmaceutical applications.

  8. Up-regulation of HB-EGF by the COX-2/PGE2 signaling associates with the cisplatin resistance and tumor recurrence of advanced HNSCC.

    Science.gov (United States)

    Yang, Cheng-Chieh; Tu, Hsi-Feng; Wu, Cheng-Hsien; Chang, Hsiu-Chuan; Chiang, Wei-Fan; Shih, Nai-Chia; Lee, Yong-Syu; Kao, Shou-Yen; Chang, Kuo-Wei

    2016-05-01

    When treating advanced HNSCC, a cisplatin-based systemic regimen benefit patient survival. However, chemoresistance will greatly reduce the effectiveness of this approach. The identification of molecules that contribute to cisplatin resistance may potentially improve the survival. Both HB-EGF and COX-2 have been reported to increase cisplatin-resistance. Here, we have focused on the regulation of HB-EGF/COX-2 and their roles in cisplatin resistance. IHC staining was used to measure the expression levels of HB-EGF and COX-2 on the tissue microarray from 43 tissue samples of patients with advanced HNSCC. siRNA, western blot and qRT-PCR were used to dissect the regulation between EGF, Akt, COX-2, PGE2, and cisplatin sensitivity. The correlation between HB-EGF, COX2 and HNSCC progression was analyzed by the receiver operating characteristic (ROC) curve and Kaplan-Meier disease free survival. Patients of advanced HNSCC patients with increased HB-EGF and COX-2 expression have higher tumor recurrent rates that was related to cisplatin resistance. The resistance was mediated via an increased expression of HB-EGF and COX-2. The activation of Akt by either EGF or areca nut extract were able to upregulate COX-2, which would increase the expression of HB-EGF in a PGE2 dependent manner. Inhibition and knockdown of COX-2 resulted in a decrease in HB-EGF. In the tissue samples from HNSCC patients, there was a significant positive correlation between the expression of COX-2 and HB-EGF. Our results suggested that COX-2 and HB-EGF are important in development of HNSCC cisplatin resistance. These findings may help the development of new strategies for overcoming cisplatin resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

    International Nuclear Information System (INIS)

    Jeong, Wooyoung; Bazer, Fuller W.; Song, Gwonhwa; Kim, Jinyoung

    2016-01-01

    The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

  10. Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Wooyoung [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of); Bazer, Fuller W. [Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A& M University, College Station, TX (United States); Song, Gwonhwa, E-mail: ghsong@korea.ac.kr [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Jinyoung, E-mail: jinyoungkim@dankook.ac.kr [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of)

    2016-01-08

    The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

  11. Epidermal growth factor and growth in vivo

    International Nuclear Information System (INIS)

    Rhodes, J.A.

    1986-01-01

    Epidermal growth factor (EGF) causes a dose-dependent thickening of the epidermis in suckling mice. The cellular mechanisms underlying this thickening were analyzed by measuring the effect of EGF on the cell-cycle. Neonatal mice were given daily injections of either 2ug EGF/g body weight/day or an equivalent volume of saline, and on the seventh day received a single injection of 3 H-thymidine. At various times the mice were perfused with fixative; 1um sections of skin were stained with a modification of Harris' hematoxylin and were autoradiographed. The sections were analyzed using three methods based on the dependence on time after injection of 3 H-thymidine of: frequency of labelled mitoses, labelling index, and reciprocal grains/nucleus. It was found that EGF caused a two-fold increase in the cell production rate. The effect of exogenous EGF on the morphology of gastric mucosa and incisors of suckling mice was also studied. The gastric mucosa appeared thicker in EGF-treated animals, but the effect was not statistically significant. In contrast to its effect on epidermis and gastric mucosa, EGF caused a significant, dose-dependent decrease in the size of the incisors. Because the mouse submandibular salivary gland is the major source of EGF the effect of sialoadenectomy on female reproductive functions was examined. Ablation of the submandibular gland had no effect on: length of estrus cycle, ability of the female to produce litters, length of the gestation period, litter size, and weight of the litter at birth. There was also no effect on survival of the offspring or on age at which the eyelids separated

  12. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    DEFF Research Database (Denmark)

    Sorkin, A; Helin, K; Waters, C M

    1992-01-01

    The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of ...

  13. Pou5f1-dependent EGF expression controls E-cad endocytosis, cell adhesion, and zebrafish epiboly movements

    Science.gov (United States)

    Song, Sungmin; Eckerle, Stephanie; Onichtchouk, Daria; Marrs, James A.; Nitschke, Roland; Driever, Wolfgang

    2013-01-01

    Summary Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that in zebrafish MZspg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cad endosomal trafficking and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGFR may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other. PMID:23484854

  14. Efectos del bloqueo del sistema EGF/EGFR sobre la cicatrización de las heridas en pacientes oncológicos

    Directory of Open Access Journals (Sweden)

    Ángel R Casacó Parada

    Full Text Available La administración de drogas que bloquean el sistema del factor de crecimiento epidérmico y su receptor ha demostrado efectos beneficiosos en pacientes con tumores sólidos de origen epitelial. Cada día resulta más frecuente el uso de múltiples modalidades terapéuticas para combatir estos tumores, las cuales incluyen la asociación de agentes blanco y cirugía. Los agentes que actúan sobre dicho sistema pudieran causar trastornos de la cicatrización al bloquear vías del sistema que también intervienen en la cicatrización de las heridas. El objetivo de este artículo es revisar y comentar acerca del conocimiento de la relación entre el uso de las drogas anti-EGF/EGFR y los trastornos en la cicatrización de las heridas. La búsqueda bibliográfica se realizó en PubMed y Google (solo en español e inglés y se tuvo en cuenta cualquier publicación encontrada hasta enero del 2014. Se incluyeron los anticuerpos monoclonales cetuximab, panitumumab y nimotuzumab; las pequeñas moléculas erlotinib y gefitinib y las vacunas terapéuticas contra el cáncer CIMAvax EGF y HER-1. Se hace especial énfasis en los biofarmacéuticos nimotuzumab, CIMAvax EGF y HER-1; producidos en el Centro de Inmunología Molecular, La Habana, Cuba, debido a su amplio uso en Cuba y otros países de América Latina. No se encontraron evidencias de relación entre el uso de estos productos y la aparición de trastornos en la cicatrización de las heridas. Dado que los tratamientos anti-EGF/EGFR también inhiben la proliferación celular que induce el drenaje de las heridas y la migración celular inducida por las radiaciones, se sugiere que el tratamiento anti-EGF/EGFR no debe suspenderse, ni antes ni después de la cirugía y sus posibles efectos deben ser vigilados. Obviamente, se necesitan ulteriores investigaciones por parte de los farmacólogos no clínicos y clínicos, oncólogos clínicos y cirujanos oncológicos para entender mejor los procesos fisiopatol

  15. Charge conjugation invariance of the spectator equations

    International Nuclear Information System (INIS)

    Gross, F.

    1999-01-01

    In response to recent criticism, the authors show how to define the spectator equations for negative energies so that charge conjugation invariance is preserved. The result, which emerges naturally from the application of spectator principles to systems of particles with negative energies, is to replace all factors of the external energies W iota by √ W 2 iota , insuring that the amplitudes are independent of the sign of the energies W iota

  16. Error Estimation in Preconditioned Conjugate Gradients

    Czech Academy of Sciences Publication Activity Database

    Strakoš, Zdeněk; Tichý, Petr

    2005-01-01

    Roč. 45, - (2005), s. 789-817 ISSN 0006-3835 R&D Projects: GA AV ČR 1ET400300415; GA AV ČR KJB1030306 Institutional research plan: CEZ:AV0Z10300504 Keywords : preconditioned conjugate gradient method * error bounds * stopping criteria * evaluation of convergence * numerical stability * finite precision arithmetic * rounding errors Subject RIV: BA - General Mathematics Impact factor: 0.509, year: 2005

  17. Epidermal growth factor regulates apoptosis and oxidative stress in a rat model of spinal cord injury.

    Science.gov (United States)

    Ozturk, Anil Murat; Sozbilen, Murat Celal; Sevgili, Elvin; Dagci, Taner; Özyalcin, Halit; Armagan, Guliz

    2018-03-22

    Spinal cord injury (SCI) leads to vascular damage and disruption of blood-spinal cord barrier which participates in secondary nerve injury. Epidermal growth factor (EGF) is an endogenous protein which regulates cell proliferation, growth and differention. Previous studies reported that EGF exerts neuroprotective effect in spinal cord after SCI. However, the molecular mechanisms underlying EGF-mediated protection in different regions of nervous system have not shown yet. In this study, we aimed to examine possible anti-apoptotic and protective roles of EGF not only in spinal cord but also in brain following SCI. Twenty-eight adult rats were divided into four groups of seven animals each as follows: sham, trauma (SCI), SCI + EGF and SCI + methylprednisolone (MP) groups. The functional neurological deficits due to the SCI were assessed by behavioral analysis using the Basso, Beattie and Bresnahan (BBB) open-field locomotor test. The alterations in pro-/anti-apoptotic protein levels and antioxidant enzyme activities were measured in spinal cord and frontal cortex. In our study, EGF promoted locomotor recovery and motor neuron survival of SCI rats. EGF treatment significantly decreased Bax and increased Bcl-2 protein expressions both in spinal cord and brain when compared to SCI group. Moreover, antioxidant enzyme activities including catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were increased following EGF treatment similar to MP treatment. Our experiment also suggests that alteration of the ratio of Bcl-2 to Bax may result from decreased apoptosis following EGF treatment. As a conclusion, these results show, for the first time, that administration of EGF exerts its protection via regulating apoptotic and oxidative pathways in response to spinal cord injury in different regions of central nervous system. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Effects of epidermal growth factor on neural crest cells in tissue culture

    International Nuclear Information System (INIS)

    Erickson, C.A.; Turley, E.A.

    1987-01-01

    Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the 3 H-labeled proteoglycan. Furthermore, EGF stimulates [ 3 H]thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis

  19. Quantitative automated microscopy (QuAM elucidates growth factor specific signalling in pain sensitization

    Directory of Open Access Journals (Sweden)

    Levine Jon D

    2010-12-01

    Full Text Available Abstract Background Dorsal root ganglia (DRG-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base. The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. Results We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+- and IB4(--neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked

  20. Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Le Thi Minh Phuc

    2017-06-01

    Full Text Available The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF on the uptake efficiency of polystyrene nanoparticles (PS NPs by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

  1. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    Energy Technology Data Exchange (ETDEWEB)

    Strom, Bjorn O. [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.no [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  2. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Velu, T; Martin, P

    1991-01-01

    mutants in the full length receptor. EGF-dependent transforming ability of the single point mutants is similar to that of the wild type, while that of double mutants is decreased and an even lower activity is present in the triple mutant. In each bioassay, including EGF-dependent focal transformation...... biologically. The EGF-R kinase activity is affected by tyrosine substitution since in vitro phosphorylation of exogenous substrates is reduced in the double and triple mutants. Autophosphorylation, in vivo and in vitro, is also reduced, but not totally abolished in the triple point mutant and Dc123 indicating......The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple...

  3. Organometallic B12-DNA conjugate

    DEFF Research Database (Denmark)

    Hunger, Miriam; Mutti, Elena; Rieder, Alexander

    2014-01-01

    Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in hum...

  4. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    International Nuclear Information System (INIS)

    Shukla, Rameshwer; Thomas, Thommey P; Desai, Ankur M; Kotlyar, Alina; Park, Steve J; Baker, James R Jr

    2008-01-01

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket

  5. Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres.

    Science.gov (United States)

    Hamdi, M M; Mutungi, G

    2010-02-01

    It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

  6. Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation.

    Science.gov (United States)

    Zhang, J; Talbot, W S; Schier, A F

    1998-01-23

    The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. We report the molecular isolation of oep using a positional cloning approach. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation.

  7. Oleic acid blocks EGF-induced [Ca2+]i release without altering cellular metabolism in fibroblast EGFR T17.

    Science.gov (United States)

    Zugaza, J L; Casabiell, X A; Bokser, L; Casanueva, F F

    1995-02-06

    EGFR-T17 cells were pretreated with oleic acid and 5-10 minutes later stimulated with EGF, to study if early ionic signals are instrumental in inducing metabolic cellular response. Oleic acid blocks EGF-induced [Ca2+]i rise and Ca2+ influx without altering 2-deoxyglucose and 2-aminobutiryc acid uptake nor acute, nor chronically. Oleic acid it is shown, in the first minutes favors the entrance of both molecules to modify the physico-chemical membrane state. On the other hand, oleic acid is unable to block protein synthesis. The results suggest that EGF-induced Ins(1,4,5)P3/Ca2+ pathway does not seem to be decisive in the control of cellular metabolic activity.

  8. GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells.

    Directory of Open Access Journals (Sweden)

    ZhenZhen Hu

    Full Text Available BACKGROUND: Epidermal growth factor (EGF signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

  9. Electrochromic in conjugated polymers

    International Nuclear Information System (INIS)

    Picado Valenzuela, Alfredo

    2007-01-01

    This revision considered object the description of one of the materials with the greatest potential in the field of electrochromic (mainly in the visible region): the conjugated polymers (CP), area of enormous potential both now and in a short time ahead. The CP are insulating materials and organic semiconductors in a state not doped. They can be doped positively or negatively being observed a significant increase in the conductivity and being generated a color change in these materials. The understanding of how optical properties vary based on the chemical structure of the polymer or its mixtures and more precisely of the alternatives that can be entered into the conjugated system or π system to obtain a material that besides to be flexible, environmentally stable, presents the colored states. The revision was centred chiefly in the polypyrrole (Ppy), the polythiophene (PTh) and their derivatives such as poly (3.4-ethylenedioxythiophene) (PEDOT). The advantage of using monomers with variable structure, to adjust the composition of the copolymer, or to blend with the PC, allows to obtain a variety of colored states that can be modulated through the visible spectrum and even with applications to wavelengths outside of this region. Because the PC presented at least two different colored states can be varied continuously as a function of the voltage applied. In some cases, they may submit multicoloured statements, which offers a range of possibilities for their application in flexible electronic devices type screens and windows. Applications include smart windows, camouflage clothing and data screens. This type of material is emerging as one of the substitutes of the traditional inorganic semiconductor, with the advantage of its low cost, high flexibility and the possibility to generate multiple colors through the handling of the monomers in the structure and control of energy of his band gap. (author) [es

  10. 131I-recombinant human EGF has antitumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

    International Nuclear Information System (INIS)

    Li Y.-C.; Xu, W.-Y.; Tan, T.-Z.; He Sheng

    2004-01-01

    This study investigated the inhibitory action of 131 I-recombinant human EGF ( 131 I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. The activity and tumor uptake of 131 I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with 131 I-rhEGF. Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. The tissue distribution assay revealed that 131 I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73%ID · g -1 ) at 120 hours at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, and 6.6-fold higher than that in blood, liver, and kidneys, respectively. Tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected 131 I-rhEGF. Tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with 131 I (7.49%) and 131 I-HSA (6.91%; P 131 I-rhEGF could significantly damage and ultimately kill tumor cells. Our results suggest that 131 I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy

  11. Influence of topical human epidermal growth factor on postkeratoplasty re-epithelialisation

    NARCIS (Netherlands)

    M.M. Dellaert; T.A. Casey; S. Wiffen; J. Gordon (Jocelynne); P. Johnson (Jürgen); A.J. Geerards (Annette); W.J. Rijneveld (Wilhelmina); L. Remeijer (Lies); W.H. Beekhuis (Houdijn); P.G.H. Mulder (Paul)

    1997-01-01

    textabstractAIM: To test the efficacy and safety of recombinant human epidermal growth factor (hEGF) on corneal re-epithelialisation following penetrating keratoplasty. METHODS: A prospective, randomised, placebo controlled study was carried out in which patients were

  12. Diabetic Foot Ulcers and Epidermal Growth Factor: Revisiting the Local Delivery Route for a Successful Outcome

    Directory of Open Access Journals (Sweden)

    Jorge Berlanga-Acosta

    2017-01-01

    Full Text Available Soon after epidermal growth factor (EGF discovery, some in vivo models appeared demonstrating its property to enhance cutaneous wound healing. EGF was the first growth factor (GF introduced in the clinical arena as a healing enhancer, exerting its mitogenic effects on epithelial, fibroblastoid, and endothelial cells via a tyrosine kinase membrane receptor. Compelling evidences from the 90s documented that, for EGF, locally prolonged bioavailability and hourly interaction with the receptor were necessary for a successful tissue response. Eventually, the enthusiasm on the clinical use of EGF to steer the healing process was wiped out as the topical route to deliver proteins started to be questioned. The simultaneous in vivo experiments, emphasizing the impact of the parenterally administered EGF on epithelial and nonepithelial organs in terms of mitogenesis and cytoprotection, rendered the theoretical fundamentals for the injectable use of EGF and shaped the hypothesis that locally infiltrating the diabetic ulcers would lead to an effective healing. Although the diabetic chronic wounds microenvironment is hostile for local GFs bioavailability, EGF local infiltration circumvented the limitations of its topical application, thus expanding its therapeutic prospect. Our clinical pharmacovigilance and basic studies attest the significance of the GF local infiltration for chronic wounds healing.

  13. Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX

    International Nuclear Information System (INIS)

    Huang, L.H.; Cheng, H.; Sweeney, W.V.; Pardi, A.; Tam, J.P.

    1991-01-01

    Factor IX is a blood clotting protein that contains three regions, including a γ-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF0like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel β-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp 28 , with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the β-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself

  14. Relationship between EGF, TGFA, and EGFR Gene Polymorphisms and Traditional Chinese Medicine ZHENG in Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Junfeng Zhang

    2013-01-01

    Full Text Available In traditional Chinese medicine (TCM, correct syndrome differentiation is the most important principle guiding the prescription of Chinese herbal formulae for the treatment of gastric cancer (GC. We aimed to reveal the genetic mechanisms underlying GC syndrome differentiation (ZHENG in a population of 387 GC patients. Twenty-nine single nucleotide polymorphisms (SNPs in EGF, TGFA, and EGFR were investigated. Two SNPs, rs11466285 in TGFA and rs884225 in EGFR, were significantly associated with the distribution of ZHENG (P<0.05. The rs11466285 TT genotype increased the risk of damp heat with toxin (DHT and deficiency of both Qi and yin (DQY compared with obstruction of blood stasis (OBS. The rs884225 AA genotype could increase the risk of DQY and deficiency of both Qi and blood (DQB compared with yin deficiency due to stomach heat (YDSH. Parallel comparison among the SNPs and syndrome types revealed that DQB was distinct from YDSH, disharmony between the liver and stomach, stagnation of phlegm muddiness (SPM, OBS, and other syndromes at several SNP loci (P<0.05. The rs11466285 TT and rs884225 AA genotypes exhibit increased risk of DQB compared with OBS and SPM (P<0.05, respectively. In conclusion, the formation of GC ZHENG was related to EGF, TGFA, and EGFR gene polymorphisms.

  15. Phase-conjugate optical coherence tomography

    International Nuclear Information System (INIS)

    Erkmen, Baris I.; Shapiro, Jeffrey H.

    2006-01-01

    Quantum optical coherence tomography (Q-OCT) offers a factor-of-2 improvement in axial resolution and the advantage of even-order dispersion cancellation when it is compared to conventional OCT (C-OCT). These features have been ascribed to the nonclassical nature of the biphoton state employed in the former, as opposed to the classical state used in the latter. Phase-conjugate OCT (PC-OCT) shows that nonclassical light is not necessary to reap Q-OCT's advantages. PC-OCT uses classical-state signal and reference beams, which have a phase-sensitive cross correlation, together with phase conjugation to achieve the axial resolution and even-order dispersion cancellation of Q-OCT with a signal-to-noise ratio that can be comparable to that of C-OCT

  16. Inhibitory effect of SPE-39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF-stimulated cells.

    Science.gov (United States)

    Ishii, Ayumi; Kamimori, Kanae; Hiyoshi, Mineyoshi; Kido, Hiroshi; Ohta, Takeshi; Konishi, Hiroaki

    2012-07-30

    Although SPE-39 is a binding protein to Vps33B that is one of the subunit in the mammalian HOPS complex, the elements of SPE-39 function remain unknown. Here, we show that tyrosine phosphorylation of SPE-39 following EGF stimulation plays a role in the stability of SPE-39 itself. Ubiquitination of the C-terminal region of SPE-39 was also elevated in response to EGF stimulation, and this process was regulated by the phosphorylation of Tyr-11 in SPE-39. However, association of Vps33B with SPE-39 inhibited the elevation of ubiquitination of SPE-39 following EGF stimulation, which might be responsible for the stabilization of SPE-39. Furthermore, an opposing functional relationship between SPE-39 and Vps33B on the downregulation of the EGF receptor was observed in EGF-stimulated COS-7 cells. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Clinical significance of measurement of changes of serum IGF-II, EGF and CYFRA21-1 levels after chemotherapy in patients with lung cancer

    International Nuclear Information System (INIS)

    Chen Jianlin

    2010-01-01

    Objective: To study the clinical significance of changes of serum IGF-II, EGF and CYFRA21-1 levels after chemotherapy in patients with lung cancer. Methods: Serum IGF-II, EGF (with RIA), and CYFRA21-1 (with ECLIA) levels were determined both before and after chemotherapy in 39 patients with lung cancer as well as once in 35 controls. Results: Before chemotherapy, serum IGF-II, EGF and CYFRA21-1 levels were significantly higher in the patients than those in controls(P<0.01). Six months after chemotherapy, serum IGF-II, EGF and CYFRA21-1 levels dropped markedly, but remained significantly higher than those in controls(P<0.05). Conclusion: The development of lung cancer in patients was closely related to the serum IGF-II, EGF and CYFRA21-1 levels. (authors)

  18. Clinical significance of measurement of changes of serum TNF-α and EGF levels after chemotherapy in patients with malignant hydatidiform mole

    International Nuclear Information System (INIS)

    Chen Xiaochao; Zhou Dongxia; Zhang Liming

    2008-01-01

    Objective: To study the clinical significance of changes of serum TNF-α and EGF levels after chemotherapy in patients with malignant hydatidiform mole. Methods: Serum TNF-α, EGF levels (with RIA) were measured both before and after chemotherapy in 32 patients with malignant hydatidiform mole as well as in 35 controls. Results: Before chemotherapy, serum TNF- α and EGF levels were significantly higher in the patients than those in controls (P<0.01). Six months after chemotherapy, serum TNF-α and EGF levels, though dropped markedly, remained significantly higher than those in controls (P<0.05). Conclusion: Development of malignant hydatidiform mole in patients was closely related to the serum TNF-α and EGF levels. (authors)

  19. Vitamin B12 Phosphate Conjugation and Its Effect on Binding to the Human B12 -Binding Proteins Intrinsic Factor and Haptocorrin

    DEFF Research Database (Denmark)

    Ó Proinsias, Keith; Ociepa, Michał; Pluta, Katarzyna

    2016-01-01

    The binding of vitamin B12 derivatives to human B12 transporter proteins is strongly influenced by the type and site of modification of the cobalamin original structure. We have prepared the first cobalamin derivative modified at the phosphate moiety. The reaction conditions were fully optimized...... and its limitations examined. The resulting derivatives, particularly those bearing terminal alkyne and azide groups, were isolated and used in copper-catalyzed alkyne-azide cycloaddition reactions (CuAAC). Their sensitivity towards light revealed their potential as photocleavable molecules. The binding...... abilities of selected derivatives were examined and compared with cyanocobalamin. The interaction of the alkylated derivatives with haptocorrin was less affected than the interaction with intrinsic factor. Furthermore, the configuration of the phosphate moiety was irrelevant to the binding process....

  20. Block-conjugate-gradient method

    International Nuclear Information System (INIS)

    McCarthy, J.F.

    1989-01-01

    It is shown that by using the block-conjugate-gradient method several, say s, columns of the inverse Kogut-Susskind fermion matrix can be found simultaneously, in less time than it would take to run the standard conjugate-gradient algorithm s times. The method improves in efficiency relative to the standard conjugate-gradient algorithm as the fermion mass is decreased and as the value of the coupling is pushed to its limit before the finite-size effects become important. Thus it is potentially useful for measuring propagators in large lattice-gauge-theory calculations of the particle spectrum

  1. A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling

    International Nuclear Information System (INIS)

    Hsieh, Ming-Chu; Hu, Wan-Ping; Yu, Hsin-Su; Wu, Wen-Chuan; Chang, Long-Sen; Kao, Ying-Hsien; Wang, Jeh-Jeng

    2011-01-01

    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. - Research highlights: → A novel carboxylate-PBD hybrid as anti-melanoma drug. → IN4CPBD interrupts melanoma cell cycle progression

  2. An evaluation of the effects of epidermal growth factor on irradiation lip mucosa damage in mice

    International Nuclear Information System (INIS)

    Feng Yan

    1994-01-01

    The effect of epidermal growth factor (EGF) on lip mucosa damage by irradiation was explored in mice. EGF was administered in doses of 100 μg/kg/day using different schedules. Mucosal damage was assessed. The metaphase arrest method with vinblastine was used to evaluate the diurnal rhythm of mitosis. EGF in regimens employed did not protect the mouse lip epithelial cells from irradiation induced damage, but it has a demonstrable stimulatory effect on cell proliferation in lip mucosa which is dependent on the schedules of administration. The reasons and mechanisms are discussed

  3. Immunohistochemical localization of epidermal growth factor in the second-trimester human fetus

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Kryger-Baggesen, N; Nexø, Ebba

    1996-01-01

    Epidermal growth factor (EGF) is considered to be important in mammalian neonatal growth and development. In order to clarify its developmental role, we have investigated, by immunohistochemistry, the localization of EGF and the time of its first appearance in various organs from a series of 25...... midtrimester human fetuses with a gestational age ranging from 13 to 22 weeks. The first detectable EGF immunoreactivity occurred in week 15-16 fetuses in the placenta, the skin, the distal tubules of the kidney, the surface epithelium of the stomach, and the tips of the small intestinal villi, as well...

  4. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    DEFF Research Database (Denmark)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  5. Combination of Gold Nanoparticle-Conjugated Tumor Necrosis Factor-α and Radiation Therapy Results in a Synergistic Antitumor Response in Murine Carcinoma Models.

    Science.gov (United States)

    Koonce, Nathan A; Quick, Charles M; Hardee, Matthew E; Jamshidi-Parsian, Azemat; Dent, Judith A; Paciotti, Giulio F; Nedosekin, Dmitry; Dings, Ruud P M; Griffin, Robert J

    2015-11-01

    Although remarkable preclinical antitumor effects have been shown for tumor necrosis factor-α (TNF) alone and combined with radiation, its clinical use has been hindered by systemic dose-limiting toxicities. We investigated the physiological and antitumor effects of radiation therapy combined with the novel nanomedicine CYT-6091, a 27-nm average-diameter polyethylene glycol-TNF-coated gold nanoparticle, which recently passed through phase 1 trials. The physiologic and antitumor effects of single and fractionated radiation combined with CYT-6091 were studied in the murine 4T1 breast carcinoma and SCCVII head and neck tumor squamous cell carcinoma models. In the 4T1 murine breast tumor model, we observed a significant reduction in the tumor interstitial fluid pressure (IFP) 24 hours after CYT-6091 alone and combined with a radiation dose of 12 Gy (P.05 vs control) despite extensive vascular damage observed. The IFP reduction in the 4T1 model was also associated with marked vascular damage and extravasation of red blood cells into the tumor interstitium. A sustained reduction in tumor cell density was observed in the combined therapy group compared with all other groups (P<.05). Finally, we observed a more than twofold delay in tumor growth when CYT-6091 was combined with a single 20-Gy radiation dose-notably, irrespective of the treatment sequence. Moreover, when hypofractionated radiation (12 Gy × 3) was applied with CYT-6091 treatment, a more than five-fold growth delay was observed in the combined treatment group of both tumor models and determined to be synergistic. Our results have demonstrated that TNF-labeled gold nanoparticles combined with single or fractionated high-dose radiation therapy is effective in reducing IFP and tumor growth and shows promise for clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Combination of Gold Nanoparticle-Conjugated Tumor Necrosis Factor-α and Radiation Therapy Results in a Synergistic Antitumor Response in Murine Carcinoma Models

    Energy Technology Data Exchange (ETDEWEB)

    Koonce, Nathan A. [Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Quick, Charles M. [Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Hardee, Matthew E.; Jamshidi-Parsian, Azemat; Dent, Judith A. [Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Paciotti, Giulio F. [CytImmune Sciences, Rockville, Maryland (United States); Nedosekin, Dmitry [Department of Otolaryngology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Dings, Ruud P.M. [Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Griffin, Robert J., E-mail: RJGriffin@uams.edu [Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States)

    2015-11-01

    Purpose: Although remarkable preclinical antitumor effects have been shown for tumor necrosis factor-α (TNF) alone and combined with radiation, its clinical use has been hindered by systemic dose-limiting toxicities. We investigated the physiological and antitumor effects of radiation therapy combined with the novel nanomedicine CYT-6091, a 27-nm average-diameter polyethylene glycol-TNF-coated gold nanoparticle, which recently passed through phase 1 trials. Methods and Materials: The physiologic and antitumor effects of single and fractionated radiation combined with CYT-6091 were studied in the murine 4T1 breast carcinoma and SCCVII head and neck tumor squamous cell carcinoma models. Results: In the 4T1 murine breast tumor model, we observed a significant reduction in the tumor interstitial fluid pressure (IFP) 24 hours after CYT-6091 alone and combined with a radiation dose of 12 Gy (P<.05 vs control). In contrast, radiation alone (12 Gy) had a negligible effect on the IFP. In the SCCVII head and neck tumor model, the baseline IFP was not markedly elevated, and little additional change occurred in the IFP after single-dose radiation or combined therapy (P>.05 vs control) despite extensive vascular damage observed. The IFP reduction in the 4T1 model was also associated with marked vascular damage and extravasation of red blood cells into the tumor interstitium. A sustained reduction in tumor cell density was observed in the combined therapy group compared with all other groups (P<.05). Finally, we observed a more than twofold delay in tumor growth when CYT-6091 was combined with a single 20-Gy radiation dose—notably, irrespective of the treatment sequence. Moreover, when hypofractionated radiation (12 Gy × 3) was applied with CYT-6091 treatment, a more than five-fold growth delay was observed in the combined treatment group of both tumor models and determined to be synergistic. Conclusions: Our results have demonstrated that TNF-labeled gold

  7. Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation

    International Nuclear Information System (INIS)

    Yarden, Y.; Schlessinger, J.

    1987-01-01

    The membrane receptor for epidermal growth factor (EGF) is a 170,000 dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane

  8. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell......Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...

  9. Entanglements in Conjugated Polymers

    Science.gov (United States)

    Xie, Renxuan; Lee, Youngmin; Aplan, Melissa; Caggiano, Nick; Gomez, Enrique; Colby, Ralph

    Conjugated polymers, such as poly(3-hexylthiophene-2,5-diyl) (P3HT) and poly-((9,9-dioctylfluorene)-2,7-diyl-alt-[4,7-bis(thiophen-5-yl)-2,1,3-benzothiadiazole]-2',2''-diyl) (PFTBT), are widely used as hole and electron transport materials in a variety of electronic devices. However, fundamental knowledge regarding chain entanglements and nematic-to-isotropic transition is still lacking and are crucial to maximize charge transport properties. A systematic melt rheology study on P3HT with various molecular weights and regio regularities was performed. We find that the entanglement molecular weight Me is 5.0 kg/mol for regiorandom P3HT, but the apparent Me for regioregular P3HT is significantly higher. The difference is postulated to arise from the presence of a nematic phase only in regioregular P3HT. Analogously, PFTBT shows a clear rheological signature of the nematic-to-isotropic transition as a reversible sharp transition at 278 C. Shearing of this nematic phase leads to anisotropic crystalline order in PFTBT. We postulate that aligning the microstructure will impact charge transport and thereby advance the field of conducting polymers. National Science Foundation.

  10. ST6Gal1, Cox-2 and HB-EGF mRNA Expression in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Aliakbar Taherian

    2015-01-01

    Full Text Available Background: ST6Gal1, Cox-2 and HB-EGF genes are involved in different tumors and their enhanced expressions often correlate with poor prognosis. In this study we assay the expressions of these genes by reverse transcriptase-PCR in 54 breast cancer samples. Methods: Tissue samples were either formalin-fixed for histopathological examination or frozen for reverse transcriptase-PCR. Image program was used for the densitometry of the image of the gels and the expression of different genes was normalized with beta actin expression. The student's t-test and correlation matrix were used for data analyses. Results: We observed significantly higher expressions of ST6Gal1 (P= 0.040, Cox- 2 (P= 0.001 and HB-EGF (P= 0.009 in the tumor region compared to the margin samples. A significant correlation was found between HB-EGF and Cox-2 expression (P= 0.001. There was a positive correlation between total score, tumor size, histology grade and nuclear grade but there was a reverse correlation between age and tumor size, histology grade and total score. Conclusion: Expressions of ST6Gal1, Cox-2 and HB-EGF in breast tumor samples in this and a number of other studies emphasize their role as important markers in breast cancer. The use of medications to inhibit either their individual expressions or the possible inhibition of all three genes may improve patient survival and prevent metastasis.

  11. Protein carriers of conjugate vaccines

    Science.gov (United States)

    Pichichero, Michael E

    2013-01-01

    The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products. PMID:23955057

  12. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  13. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  14. The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

    Science.gov (United States)

    Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

    2000-04-01

    Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

  15. Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

    Science.gov (United States)

    Opgenorth, A; Nation, N; Graham, K; McFadden, G

    1993-02-01

    The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

  16. Effect of pre- and postpartum supplementation with lipid-encapsulated conjugated linoleic acid on reproductive performance and the growth hormone-insulin-like growth factor-I axis in multiparous high-producing dairy cows.

    Science.gov (United States)

    Csillik, Z; Faigl, V; Keresztes, M; Galamb, E; Hammon, H M; Tröscher, A; Fébel, H; Kulcsár, M; Husvéth, F; Huszenicza, Gy; Butler, W R

    2017-07-01

    The objective of the study was to evaluate the effect of prepartum and postpartum (PP) supplementation with 2 isomers of conjugated linoleic acid (CLA) on reproductive parameters and some related metabolic factors in dairy cows. High-producing, multiparous Holstein Friesian cows (n = 60) were allotted to 3 treatment groups: the CLA1 group (n = 20) was supplemented with 70 g of lipid-encapsulated CLA providing 7 g each of cis-9,trans-11 and trans-10,cis-12 CLA from d 21 (d 21) before expected calving until d 7 after artificial insemination (AI), that is, until 77 to 91 d PP; the CLA2 group (n = 20) was supplemented with the same amount of CLA beginning at calving until d 7 after AI; and the control group (n = 20) received an isocaloric, isonitrogenous, and isolipidic diet. Blood samples were taken weekly to measure glucose, insulin, insulin-like growth factor-I (IGF-I), and leptin. Liver biopsy was performed in 10 cows per group for growth hormone receptor 1A and IGF-I mRNA analyses. At d 49 to 63 PP, ovulation was synchronized with the Pre-Synch protocol followed by fixed-time AI. Milk progesterone was monitored from calving until d 35 post-AI. Cows returning to estrus following AI were inseminated. Supplementation with CLA before calving improved the recovery of plasma leptin levels in the early PP period (from the day of calving until wk 3 PP; treatment effect). Later PP (wk 5), plasma IGF-I, and leptin remained significantly higher in both CLA1 and CLA2 groups compared with control, although hepatocellular IGF-I mRNA was not different among groups. Plasma IGF-I levels remained higher in both CLA-treated groups on the day of AI. Growth hormone receptor 1A mRNA levels in hepatic tissue decreased in all groups, reaching a nadir in the first week PP. Days to first PP ovulation did not differ between groups; however, both supplemented groups conceived earlier compared with control (d 97 ± 19, d 97 ± 23, and d 113 ± 30 for CLA1, CLA2, and control, respectively

  17. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei

    2015-11-16

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  18. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    Science.gov (United States)

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

  19. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  20. Conjugated Fatty Acid Synthesis

    Science.gov (United States)

    Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

    2012-01-01

    Conjugated linolenic acids (CLNs), 18:3 Δ9,11,13, lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ9,12,15). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ9cis,11trans,13cis) or α-eleostearic acid (18:3 Δ9cis,11trans,13trans). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation. PMID:22451660

  1. Tissue remodeling induced by hypersecreted epidermal growth factor and amphiregulin in the airway after an acute asthma attack.

    Science.gov (United States)

    Enomoto, Yukinori; Orihara, Kanami; Takamasu, Tetsuya; Matsuda, Akio; Gon, Yasuhiro; Saito, Hirohisa; Ra, Chisei; Okayama, Yoshimichi

    2009-11-01

    Epidermal growth factor receptor ligands, such as epidermal growth factor (EGF) and amphiregulin, may play key roles in tissue remodeling in asthma. However, the kinetics of EGF and amphiregulin secretion in the airway after an acute asthma attack and the effect of prolonged airway exposure to these ligands on airway remodeling are unknown. To measure the EGF and amphiregulin concentrations in sputa obtained from patients with asthma under various conditions, and to examine the effects of EGF and amphiregulin on the proliferation or differentiation of airway structural cells. Epidermal growth factor and amphiregulin levels were measured by ELISA in sputum specimens collected from 14 hospitalized children with asthma during an acute asthma attack, 13 stable outpatients with asthma, 8 healthy control children, and 7 children with respiratory tract infections. The effects of EGF and amphiregulin on the proliferation and/or differentiation of normal human bronchial epithelial cells (NHBE), bronchial smooth muscle cells (BSMC), and normal human lung fibroblasts (NHLF) were examined. The sputum levels of EGF were significantly higher for about a week after an acute asthma attack compared with the levels in stable subjects with asthma and control subjects. In contrast, upregulation of amphiregulin in the sputa of patients with asthma was observed only during the acute attack. EGF caused proliferation of NHBE, BSMC, and NHLF, whereas amphiregulin induced proliferation of only NHBE. Prolonged exposure of NHBE to EGF and amphiregulin induced mucous cell metaplasia in an IL-13-independent manner. Acute asthma attacks are associated with hypersecretion of EGF and amphiregulin in the airway. Recurrent acute attacks may aggravate airway remodeling.

  2. Comparison of the pharmacological and biological properties of HPMA copolymer-pirarubicin conjugates: A single-chain copolymer conjugate and its biodegradable tandem-diblock copolymer conjugate

    Czech Academy of Sciences Publication Activity Database

    Etrych, Tomáš; Tsukigawa, K.; Nakamura, H.; Chytil, Petr; Fang, J.; Ulbrich, Karel; Otagiri, M.; Maeda, H.

    2017-01-01

    Roč. 106, 30 August (2017), s. 10-19 ISSN 0928-0987 R&D Projects: GA ČR(CZ) GA15-02986S; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:AV ČR,Japan Society for the Promotion of Science(CZ) JSPS-16-05 Program:Bilaterální spolupráce Institutional support: RVO:61389013 Keywords : pirarubicin * PHPMA conjugate * tandem-diblock PHPMA conjugate Subject RIV: FR - Pharmacology ; Medidal Chemistry OBOR OECD: Pharmacology and pharmacy Impact factor: 3.756, year: 2016

  3. 131I-Recombinant human EGF has anti-tumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

    International Nuclear Information System (INIS)

    Li Yunchun; Tan Tianzhi; Xu Weiyun; He Sheng

    2004-01-01

    Purpose: This study investigated the inhibitory action of 131 I-recombinant human EGF ( 131 I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. Methods: The activity and tumor uptake of 131 I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with 131 I-rhEGF, Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. Results: The tissue distribution assay revealed that 131 I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73% ID·g-l) at 120 h, at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, 6.6-fold higher than that in blood, liver, kidneys, respectively. The tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected 131 I-rhEGF. The extent of tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with 131 I (7.49%) and 131 I-HSA (6.91%; P 131 I-rhEGF could significantly damage and ultimately kill tumor cells. Conclusions: Our results suggest that 131 I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy. Key words. Iodine-131 rhEGF Breast cancer Therapy. (authors)

  4. Model of a ternary complex between activated factor VII, tissue factor and factor IX.

    Science.gov (United States)

    Chen, Shu-wen W; Pellequer, Jean-Luc; Schved, Jean-François; Giansily-Blaizot, Muriel

    2002-07-01

    Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

  5. A fast, preconditioned conjugate gradient Toeplitz solver

    Science.gov (United States)

    Pan, Victor; Schrieber, Robert

    1989-01-01

    A simple factorization is given of an arbitrary hermitian, positive definite matrix in which the factors are well-conditioned, hermitian, and positive definite. In fact, given knowledge of the extreme eigenvalues of the original matrix A, an optimal improvement can be achieved, making the condition numbers of each of the two factors equal to the square root of the condition number of A. This technique is to applied to the solution of hermitian, positive definite Toeplitz systems. Large linear systems with hermitian, positive definite Toeplitz matrices arise in some signal processing applications. A stable fast algorithm is given for solving these systems that is based on the preconditioned conjugate gradient method. The algorithm exploits Toeplitz structure to reduce the cost of an iteration to O(n log n) by applying the fast Fourier Transform to compute matrix-vector products. Matrix factorization is used as a preconditioner.

  6. Research study of conjugate materials; Conjugate material no chosa kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-03-01

    The paper reported an introductory research on possibilities of new glass `conjugate materials.` The report took up the structure and synthetic process of conjugate materials to be researched/developed, classified them according to structural elements on molecular, nanometer and cluster levels, and introduced the structures and functions. Further, as glasses with new functions to be proposed, the paper introduced transparent and high-strength glass used for houses and vehicles, light modulation glass which realizes energy saving and optical data processing, and environmentally functional glass which realizes environmental cleaning or high performance biosensor. An initial survey was also conducted on rights of intellectual property to be taken notice of in Japan and abroad in the present situation. Reports were summed up and introduced of Osaka National Research Institute, Electrotechnical Laboratory, and National Industrial Research Institute of Nagoya which are all carrying out leading studies of conjugate materials. 235 refs., 135 figs., 6 tabs.

  7. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Jarett, L.

    1990-01-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity

  8. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  9. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    Science.gov (United States)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  10. Conjugated polymer nanoparticles, methods of using, and methods of making

    KAUST Repository

    Habuchi, Satoshi

    2017-03-16

    Embodiments of the present disclosure provide for conjugated polymer nanoparticle, method of making conjugated polymer nanoparticles, method of using conjugated polymer nanoparticle, polymers, and the like.

  11. Conjugated polymer nanoparticles, methods of using, and methods of making

    KAUST Repository

    Habuchi, Satoshi; Piwonski, Hubert Marek; Michinobu, Tsuyoshi

    2017-01-01

    Embodiments of the present disclosure provide for conjugated polymer nanoparticle, method of making conjugated polymer nanoparticles, method of using conjugated polymer nanoparticle, polymers, and the like.

  12. Carboxy terminal peptide of type I procollagen and epidermal growth factor in patients with different viral hepatitis

    International Nuclear Information System (INIS)

    Han Lihong; Gong Shoujun; Li Guangming; Li Yebing; Xu Bin

    2001-01-01

    The author observed the serum levels of carboxy terminal peptide of type I procollagen (PICP) and epidermal growth factor (EGF) in the patients with viral hepatitis and cirrhosis. The serum PICP and EGF were detected in 164 cases by RIA. The results indicated that two indexes increased significantly in patients with severe chronic hepatitis, chronic persistent hepatitis and post hepatitis cirrhosis compared with normal control (P 0.05). The results showed that detection of serum PICP and EGF may be valuable diagnostic markers to assess the degree of liver inflammation and fibrosis in viral liver diseases

  13. Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

    OpenAIRE

    Matsumoto, M; Imagawa, M; Aoki, Y

    2000-01-01

    Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did no...

  14. Substituted Polyacetylenes Prepared with Rh Catalysts: From Linear to Network-Type Conjugated Polymers

    Czech Academy of Sciences Publication Activity Database

    Sedláček, J.; Balcar, Hynek

    2017-01-01

    Roč. 57, č. 1 (2017), s. 31-51 ISSN 1558-3724 Institutional support: RVO:61388955 Keywords : conjugated polymers * polyacetylenes * conjugated polymer networks Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Polymer science Impact factor: 6.459, year: 2016

  15. Conjugate Gradient like methods and their application to fixed source neutron diffusion problems

    International Nuclear Information System (INIS)

    Suetomi, Eiichi; Sekimoto, Hiroshi

    1989-01-01

    This paper presents a number of fast iterative methods for solving systems of linear equations appearing in fixed source problems for neutron diffusion. We employed the conjugate gradient and conjugate residual methods. In order to accelerate the conjugate residual method, we proposed the conjugate residual squared method by transforming the residual polynomial of the conjugate residual method. Since the convergence of these methods depends on the spectrum of coefficient matrix, we employed the incomplete Choleski (IC) factorization and the modified IC (MIC) factorization as preconditioners. These methods were applied to some neutron diffusion problems and compared with the successive overrelaxation (SOR) method. The results of these numerical experiments showed superior convergence characteristics of the conjugate gradient like method with MIC factorization to the SOR method, especially for a problem involving void region. The CPU time of the MICCG, MICCR and MICCRS methods showed no great difference. In order to vectorize the conjugate gradient like methods based on (M)IC factorization, the hyperplane method was used and implemented on the vector computers, the HITAC S-820/80 and ETA10-E (one processor mode). Significant decrease of the CPU times was observed on the S-820/80. Since the scaled conjugate gradient (SCG) method can be vectorized with no manipulation, it was also compared with the above methods. It turned out the SCG method was the fastest with respect to the CPU times on the ETA10-E. These results suggest that one should implement suitable algorithm for different vector computers. (author)

  16. Conjugated polymer zwitterions and solar cells comprising conjugated polymer zwitterions

    Science.gov (United States)

    Emrick, Todd; Russell, Thomas; Page, Zachariah; Liu, Yao

    2018-06-05

    A conjugated polymer zwitterion includes repeating units having structure (I), (II), or a combination thereof ##STR00001## wherein Ar is independently at each occurrence a divalent substituted or unsubstituted C3-30 arylene or heteroarylene group; L is independently at each occurrence a divalent C1-16 alkylene group, C6-30arylene or heteroarylene group, or alkylene oxide group; and R1 is independently at each occurrence a zwitterion. A polymer solar cell including the conjugated polymer zwitterion is also disclosed.

  17. Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

    Science.gov (United States)

    Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

    2011-04-01

    The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  18. Tangeretin and its metabolite 4′-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K

    Science.gov (United States)

    Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, MR

    2011-01-01

    BACKGROUND AND PURPOSE The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4′-hydroxy-5,6,7,8-tetramethoxyflavone (4′-OH-TMF) in this effect. EXPERIMENTAL APPROACH We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [3H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. KEY RESULTS Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4′-OH-TMF. Low micromolar concentrations of tangeretin or 4′-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4′-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. CONCLUSIONS AND IMPLICATIONS Tangeretin and 4′-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4′-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. PMID:21198542

  19. 重组人表皮生长因子对植皮创面成活的影响%Effect of recombinant human epithelium growth factor on livability of skin graft

    Institute of Scientific and Technical Information of China (English)

    龙剑虹; 张明华; 谢庭鸿; 杨兴华; 黄晓元; 周捷

    2003-01-01

    AIM: To investigate the effects of recombinant human epithelium growth factor (rhEGF) applied to skin graft. METHODS: 96 cases between February 2000 and December 2001, were treated. During the operation, After scar removed and skin grafted, the rhEGF was injected under the skin graft. 80 cases without injection of rhEGF were made as contrast. Ten days later, the area of survived skin was measured and the livability of skin was calculated. RESULTS: The skin livability of cases with injection of rhEGF was (90.67 ± 10.02)% and the skin livability of contrast cases was(76. 85 ± 8.35)%. There axisted evident differences between them( P < 0. 01) . CONCLUSION: The rhEGF was an effective method for increasing livability of skin graft.

  20. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation.

    Directory of Open Access Journals (Sweden)

    Ching Chang Cho

    Full Text Available The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs.

  1. Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Nordberg, Erika; Steffen, Ann-Charlott; Sundberg, Aasa L.; Carlsson, Joergen [Uppsala University, Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Persson, Mikael [Uppsala University, Division of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Uppsala University, Division of Experimental Urology, Department of Surgical Sciences, Rudbeck Laboratory, Uppsala (Sweden); Glimelius, Bengt [Uppsala University, Division of Oncology, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden)

    2005-07-01

    The purpose of this study was to analyse whether non-radiolabelled epidermal growth factor (EGF) can modify the cellular uptake of {sup 125}I when delivered as [{sup 125}I]trastuzumab. {sup 125}I was used as a marker for the diagnostically and therapeutically more interesting isotopes {sup 123}I (SPECT), {sup 124}I (PET) and {sup 131}I (therapy). The cell-associated radioactivity was measured in squamous carcinoma A431 cells following addition of [{sup 125}I]trastuzumab. Different concentrations of [{sup 125}I]trastuzumab and unlabelled EGF were used, and the total, membrane-bound and internalised radioactivity was measured. We also analysed how EGF and trastuzumab affected the cell growth. It was generally found that the cellular {sup 125}I uptake was decreased by the addition of EGF when [{sup 125}I]trastuzumab was added for short incubation times. However, if the incubation times were longer, EGF increased the {sup 125}I uptake. This shift came earlier when higher [{sup 125}I]trastuzumab concentrations were applied. The addition of EGF also influenced cell proliferation, and concentrations above 10 ng/ml reduced cell growth by approximately 20% after 24 h of incubation. By adding unlabelled EGF, it was possible to modify the cellular uptake of [{sup 125}I]trastuzumab. This points towards new approaches for the modification of radionuclide uptake in EGFR- and HER2-positive tumours. (orig.)

  2. 125I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    International Nuclear Information System (INIS)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of 125 I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class A/B diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more 125 I-hEGF than did fetal membranes (P 125 I-hEGF binding to fetal membranes from the various pregnancy states (P 125 I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P 125 I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P 125 I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P 125 I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone. (author)

  3. Effects of epidermal growth factor in artificial tear on vitamin C levels of corneal wounded eye tissues.

    Science.gov (United States)

    Gönül, B; Kaplan, B; Bilgihan, K; Budak, M T

    2001-04-01

    To investigate the effect of artificial tear (AT) solution and epidermal growth factor (EGF) treatment on the cornea and aqueous humour ascorbic acid (AA) levels of full-thickness corneal wounded eyes. The effect of EGF on the AA levels of aqueous humour and corneal wound tissue was determined in full-thickness corneal wounded rabbit eyes on the seventh post-operative day. There were three groups: untreated controls, AT-treated controls and an EGF+AT-treated experimental group (n = 6 in each group). Corneal wounded eyes were topically treated with 5 microl AT or 5 microl EGF in AT (1 mg/l EGF in AT prepaaration which contained 3.0% carbopol 940) twice daily for 6 days after operation. The wound strengths were also measured on the seventh post-operative day as a measure of wound healing. Statistical analysis was carried out using the Mann-Whitney U-test by Statview program. The wound strengths of corneas, and AA levels of wound tissues and aqueous humour, increased significantly following AT and EGF treatment (p < 0.05). In the corneal wounded eye, aqueous humour serves as a source of vitamin C and there may be a relation between EGF treatment in AT and AA levels of corneal wounded eye tissues.

  4. Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands

    International Nuclear Information System (INIS)

    Vonderhaar, B.K.; Tang, E.; Lyster, R.R.; Nascimento, M.C.

    1986-01-01

    The specific binding of iodinated epidermal growth factor ([ 125 I]iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites [dissociation constant (Kd) = 0.7-1.8 nM]. Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status

  5. Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor α to epidermal growth factor receptors promotes cell proliferation

    International Nuclear Information System (INIS)

    Anklesaria, P.; Greenberger, J.S.; Teixido, J.; Laiho, M.; Massague, J.; Pierce, J.H.

    1990-01-01

    The precursor for transforming growth factor α, pro-TGF-α, is a cell surface glycoprotein that can establish contact with epidermal growth factor (EGF) receptors on adjacent cells. To examine whether the pro-TGF-α/EGF receptor pair can simultaneously mediate cell adhesion and promote cell proliferation, the authors have expressed pro-TGF-α in a bone marrow stromal cell line labeled with [ 35 S] cysteine. Expression of pro-TGF-α allows these cells to support long-term attachment of an EGF/interleukin-3-dependent hematopoietic progenitor cell line that expresses EGF receptors but is unable to adhere to normal stroma. This interaction is inhibited by soluble EGF receptor ligands. Further, the hematopoietic progenitor cells replicate their DNA while they are attached to the stromal cell layer and become foci of sustained cell proliferation. Thus, pro-TGF-α and the EGF receptor can function as mediators of intercellular adhesion and this interaction may promote a mitogenic response. They propose the term juxtacrine to designate this form of stimulation between adjacent cells

  6. [Effect of total flavones from Cuscuta chinensis on expression of Fas/FasL, PCNA and HB-EGF in SD rats model with bromocriptine-induced abortion].

    Science.gov (United States)

    Ma, Hong-Xia; You, Zhao-Ling; Wang, Xiao-Yun

    2008-11-01

    To explore the effect of total flavones from cuscuta chinensis (TFCC) on expression of Fas, PCNA and HB-EGF in SD rats model with bromocriptine-induced abortion. The model rats of bromocriptine during 6-8 d of pregnancy induced early abortion was established, adopting respectively herbs in high and low dosage and progesterone affect model rat and after 12 d, Immunohistochemical was applied to determine Fas, HB-EGF and PCNA in deciduas and placenta. Expression of PCNA on trophoblast and deciduas, HB-EGF on trophoblast, PR on deciduas in the model used Semen cuscutae flavonoid, proesterone and normal pregnacy, were significantlly higher than those of the pure model. Expression of Fas on trophoblast and deciduas in above four groups, were significantlly lower than those of the pure model. There were no expression of HB-EGF on deciduas. TFCC regulates the proliferation and apoptosis of the deciduas and cytotrophoblasts and prevents spontaneous abortions.

  7. Evaluation of mutagenic/antimutagenic activity of conjugated linoleic ...

    African Journals Online (AJOL)

    Jane

    2011-10-12

    Oct 12, 2011 ... INTRODUCTION. Conjugated linoleic acid (CLA) is a dietary adjuvant for its ... who found unidentified anticarcinogenic factors in fried ground beef. ..... products decreases rectum cancer in 13% and colon cancer in 34%. .... alters mammary gland morphogenesis and reduces cancer risk in rats. J. Nutr.

  8. Accurate reanalysis of structures by a preconditioned conjugate gradient method

    Czech Academy of Sciences Publication Activity Database

    Kirsch, U.; Kočvara, Michal; Zowe, J.

    2002-01-01

    Roč. 55, č. 2 (2002), s. 233-251 ISSN 0029-5981 R&D Projects: GA AV ČR IAA1075005 Grant - others:BMBF(DE) 03ZOM3ER Institutional research plan: CEZ:AV0Z1075907 Keywords : preconditioned conjugate gradient s * structural reanalysis Subject RIV: BA - General Mathematics Impact factor: 1.468, year: 2002

  9. High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

    International Nuclear Information System (INIS)

    Auf, Gregor; Vajkoczy, Peter; Seno, Masaharu; Bikfalvi, Andreas; Minchenko, Dmitri; Minchenko, Oleksandr; Moenner, Michel; Jabouille, Arnaud; Delugin, Maylis; Guérit, Sylvaine; Pineau, Raphael; North, Sophie; Platonova, Natalia; Maitre, Marlène; Favereaux, Alexandre

    2013-01-01

    Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the

  10. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue

    2006-12-01

    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  11. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice.

    Directory of Open Access Journals (Sweden)

    Alexei Shir

    2006-01-01

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC], a strong activator of apoptosis, selectively to cancer cells. METHODS AND FINDINGS: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF. EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. CONCLUSION: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

  12. Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

    Science.gov (United States)

    Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

    2013-01-01

    Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

  13. Conjugal Pairing in Escherichia Coli

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 8. Conjugal Pairing in Escherichia Coli. Joshua Lederberg. Classics Volume 13 Issue 8 August 2008 pp 793-794. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/013/08/0793-0794 ...

  14. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the suc...

  15. Orderings for conjugate gradient preconditionings

    Science.gov (United States)

    Ortega, James M.

    1991-01-01

    The effect of orderings on the rate of convergence of the conjugate gradient method with SSOR or incomplete Cholesky preconditioning is examined. Some results also are presented that help to explain why red/black ordering gives an inferior rate of convergence.

  16. REVIEW ARTICLE Conjugated Hyperbilirubinaemia in Early Infancy ...

    African Journals Online (AJOL)

    REVIEW ARTICLE Conjugated Hyperbilirubinaemia in Early Infancy. AOK Johnson. Abstract. Conjugated hyperbilirubinaemia exists when the conjugated serum bilirubin level is more than 2 mg/dl or more than 20 per cent of the total serum bilirubin. It is always pathological in early infancy. The causes are many and diverse ...

  17. Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

    Science.gov (United States)

    Yunus, Ali A.; Lima, Christopher D.

    2009-01-01

    SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

  18. Effects of growth-promoting factors on proliferation of mouse ...

    African Journals Online (AJOL)

    SSCs) in vitro are critical to our understanding of male infertility, genetic resources and endangered species conservation. To investigate the effects of growth-promoting factors, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and ...

  19. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  20. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    International Nuclear Information System (INIS)

    Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-01-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  1. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ian G Woods

    2005-03-01

    Full Text Available Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

  2. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection

    International Nuclear Information System (INIS)

    Yoneda, T.; Urade, M.; Sakuda, M.; Miyazaki, T.

    1986-01-01

    We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of 125 I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound 125 I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients

  3. Polyamine conjugates of stigmasterol

    Czech Academy of Sciences Publication Activity Database

    Vida, N.; Svobodová, Hana; Rárová, L.; Drašar, P.; Šaman, David; Cvačka, Josef; Wimmer, Zdeněk

    2012-01-01

    Roč. 77, č. 12 (2012), s. 1212-1218 ISSN 0039-128X R&D Projects: GA ČR(CZ) GAP503/11/0616; GA MŠk(CZ) OC10001 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z40550506 Keywords : polyamine * stigmasterol * amide Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 2.803, year: 2012

  4. Singlet Exciton Lifetimes in Conjugated Polymer Films for Organic Solar Cells

    KAUST Repository

    Dimitrov, Stoichko; Schroeder, Bob; Nielsen, Christian; Bronstein, Hugo; Fei, Zhuping; McCulloch, Iain; Heeney, Martin; Durrant, James

    2016-01-01

    The lifetime of singlet excitons in conjugated polymer films is a key factor taken into account during organic solar cell device optimization. It determines the singlet exciton diffusion lengths in polymer films and has a direct impact

  5. Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome.

    Science.gov (United States)

    Cui, Xiu-Fang; Zhou, Wei-Mei; Yang, Yan; Zhou, Jun; Li, Xue-Liang; Lin, Lin; Zhang, Hong-Jie

    2014-10-07

    To investigate the role of epidermal growth factor (EGF) in visceral hypersensitivity and its effect on the serotonin transporter (SERT). A rat model for visceral hypersensitivity was established by intra-colonic infusion of 0.5% acetic acid in 10-d-old Sprague-Dawley rats. The visceral sensitivity was assessed by observing the abdominal withdrawal reflex and recording electromyographic activity of the external oblique muscle in response to colorectal distension. An enzyme-linked immunosorbent assay was used to measure the EGF levels in plasma and colonic tissues. SERT mRNA expression was detected by real-time PCR while protein level was determined by Western blot. The correlation between EGF and SERT levels in colon tissues was analyzed by Pearson's correlation analysis. SERT function was examined by tritiated serotonin (5-HT) uptake experiments. Rat intestinal epithelial cells (IEC-6) were used to examine the EGF regulatory effect on SERT expression and function via the EGF receptor (EGFR). EGF levels were significantly lower in the rats with visceral hypersensitivity as measured in plasma (2.639 ± 0.107 ng/mL vs 4.066 ± 0.573 ng/mL, P < 0.01) and in colonic tissue (3.244 ± 0.135 ng/100 mg vs 3.582 ± 0.197 ng/100 mg colon tissue, P < 0.01) compared with controls. Moreover, the EGF levels were positively correlated with SERT levels (r = 0.820, P < 0.01). EGF displayed dose- and time-dependent increased SERT gene expressions in IEC-6 cells. An EGFR kinase inhibitor inhibited the effect of EGF on SERT gene upregulation. SERT activity was enhanced following treatment with EGF (592.908 ± 31.515 fmol/min per milligram vs 316.789 ± 85.652 fmol/min per milligram protein, P < 0.05) and blocked by the EGFR kinase inhibitor in IEC-6 cells (590.274 ± 25.954 fmol/min per milligram vs 367.834 ± 120.307 fmol/min per milligram protein, P < 0.05). A decrease in EGF levels may contribute to the formation of visceral hypersensitivity through downregulation of SERT

  6. Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    Directory of Open Access Journals (Sweden)

    Miquella G. Chavez

    2012-01-01

    Full Text Available Modulation of cell : cell junctions is a key event in cutaneous wound repair. In this study we report that activation of the epidermal growth factor (EGF receptor disrupts cel : cell adhesion, but with different kinetics and fates for the desmosomal cadherin desmoglein and for E-cadherin. Downregulation of desmoglein preceded that of E-cadherin in vivo and in an EGF-stimulated in vitro wound reepithelialization model. Dual immunofluorescence staining revealed that neither E-cadherin nor desmoglein-2 internalized with the EGF receptor, or with one another. In response to EGF, desmoglein-2 entered a recycling compartment based on predominant colocalization with the recycling marker Rab11. In contrast, E-cadherin downregulation was accompanied by cleavage of the extracellular domain. A broad-spectrum matrix metalloproteinase inhibitor protected E-cadherin but not the desmosomal cadherin, desmoglein-2, from EGF-stimulated disruption. These findings demonstrate that although activation of the EGF receptor regulates adherens junction and desmosomal components, this stimulus downregulates associated cadherins through different mechanisms.

  7. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  8. Fisetin inhibits epidermal growth factor-induced migration of ARPE-19 cells by suppression of AKT activation and Sp1-dependent MMP-9 expression.

    Science.gov (United States)

    Lin, Hung-Yu; Chen, Yong-Syuan; Wang, Kai; Chien, Hsiang-Wen; Hsieh, Yi-Hsien; Yang, Shun-Fa

    2017-01-01

    Proliferative vitreoretinopathy (PVR) can result in abnormal migration of RPE cells. Fisetin is a naturally occurring compound that has been reported to have antitumor effects, but its effects on epidermal growth factor (EGF)-induced cell migration and the underlying mechanisms remain unclear. Effects of fisetin on EGF-induced cell viability and migration were examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in vitro migration assays. Reverse transcription-PCR (RT-PCR) and immunoblotting were performed to evaluate matrix metallopeptidase-9 (MMP-9) expression and activation of specificity protein-1 (Sp1) and protein kinase B (AKT) in ARPE-19 cells treated with EGF and with or without fisetin. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to examine Sp1 transcription activity and MMP-9 binding activity. Fisetin did not affect ARPE-19 cell viability and significantly inhibited the EGF-induced migration capacity of ARPE-19 cells. Furthermore, fisetin exerted an antimigratory effect and suppressed MMP-9 mRNA and protein expression. Treatment with EGF induced phosphorylation of AKT and expression of MMP-9 and Sp1. Fisetin combined with LY294002 (an inhibitor of AKT) prevented the EGF-induced migration involved in downregulation of Sp1 and MMP-9 expression. Luciferase and ChIP assays suggested that fisetin remarkably decreased the EGF-induced transcription activity of MMP-9 and Sp1 and inhibited EGF-mediated Sp1 from directly binding to the MMP-9 promoter in ARPE-19 cells. Fisetin inhibited EGF-induced cell migration via modulation of AKT/Sp1-dependent MMP-9 transcriptional activity. Therefore, fisetin may be a potential agent in the treatment of migratory PVR diseases.

  9. Biochemical and ultrastructural processing of [125I]epidermal growth factor in rat epidermis and hair follicles: accumulation of nuclear label

    International Nuclear Information System (INIS)

    Green, M.R.; Mycock, C.; Smith, C.G.; Couchman, J.R.

    1987-01-01

    Although the intracellular ultrastructural processing of epidermal growth factor (EGF) and its receptor have been described in cell culture systems, very few studies have examined this phenomenon in intact tissues. We have examined the ultrastructural and biochemical handling of [ 125 I]EGF in the epidermis and hair follicle bulb of intact, viable, 3- to 5-day-old rat skin the EGF receptor distribution of which has already been documented and in which EGF has been shown to be biologically active. After incubation of explants with 10 nM [ 125 I]EGF for 2.5 h at 25 degrees or 37 degrees C, radiolabel was detected over the basal cells of the epidermis and hair follicle outer root sheath, confirming previous light microscope observations. More specifically, silver grains were observed near coated and uncoated plasma membrane and coated membrane invaginations, Golgi apparatus, lysosomal structures, and nuclei. Sodium azide inhibited internalization of label, whereas a series of lysosomal inhibitors (chloroquine, monensin, and iodoacetamide) caused a slight increase in silver grains associated with lysosomal vesicles and a decrease in nuclear label. Biochemical analysis indicated that greater than 35% of radioactivity following incubation at 37 degrees C was in the form of degraded [ 125 I]EGF fragments and that inclusion of chloroquine, monensin, and iodoacetamide reduced this value to 20.8%, 8.6%, and 4.0%, respectively. In addition, chloramine T-prepared [ 125 I]EGF was found to be covalently cross-linked with low efficiency to a protein having the molecular weight of the EGF receptor. These data are discussed in the light of the effects of EGF on epithelial cell proliferation in skin

  10. Studing Regional Wave Source Time Functions Using A Massive Automated EGF Deconvolution Procedure

    Science.gov (United States)

    Xie, J. "; Schaff, D. P.

    2010-12-01

    Reliably estimated source time functions (STF) from high-frequency regional waveforms, such as Lg, Pn and Pg, provide important input for seismic source studies, explosion detection, and minimization of parameter trade-off in attenuation studies. The empirical Green’s function (EGF) method can be used for estimating STF, but it requires a strict recording condition. Waveforms from pairs of events that are similar in focal mechanism, but different in magnitude must be on-scale recorded on the same stations for the method to work. Searching for such waveforms can be very time consuming, particularly for regional waves that contain complex path effects and have reduced S/N ratios due to attenuation. We have developed a massive, automated procedure to conduct inter-event waveform deconvolution calculations from many candidate event pairs. The procedure automatically evaluates the “spikiness” of the deconvolutions by calculating their “sdc”, which is defined as the peak divided by the background value. The background value is calculated as the mean absolute value of the deconvolution, excluding 10 s around the source time function. When the sdc values are about 10 or higher, the deconvolutions are found to be sufficiently spiky (pulse-like), indicating similar path Green’s functions and good estimates of the STF. We have applied this automated procedure to Lg waves and full regional wavetrains from 989 M ≥ 5 events in and around China, calculating about a million deconvolutions. Of these we found about 2700 deconvolutions with sdc greater than 9, which, if having a sufficiently broad frequency band, can be used to estimate the STF of the larger events. We are currently refining our procedure, as well as the estimated STFs. We will infer the source scaling using the STFs. We will also explore the possibility that the deconvolution procedure could complement cross-correlation in a real time event-screening process.

  11. FGF2 and EGF induce epithelial-mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal.

    Science.gov (United States)

    Schelch, Karin; Wagner, Christina; Hager, Sonja; Pirker, Christine; Siess, Katharina; Lang, Elisabeth; Lin, Ruby; Kirschner, Michaela B; Mohr, Thomas; Brcic, Luka; Marian, Brigitte; Holzmann, Klaus; Grasl-Kraupp, Bettina; Krupitza, Georg; Laszlo, Viktoria; Klikovits, Thomas; Dome, Balazs; Hegedus, Balazs; Garay, Tamas; Reid, Glen; van Zandwijk, Nico; Klepetko, Walter; Berger, Walter; Grusch, Michael; Hoda, Mir Alireza

    2018-04-05

    Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFβ or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.

  12. Production of transforming growth factor α in human pancreatic cancer cells: evidence for a superagonist autocrine cycle

    International Nuclear Information System (INIS)

    Smith, J.J.; Derynck, R.; Korc, M.

    1987-01-01

    Previous work showed that cultured human pancreatic cancer cells overexpress the epidermal growth factor (EGF) receptor. In the present study, the authors sought to determine whether some of these cell lines produce transforming growth factor α (TGF-α). Utilizing a radiolabeled TGF-α cDNA in hybridization experiments, they determined that ASPC-1, T 3 M 4 , PANC-1, COLO-357, and MIA PaCa-2 cell lines expressed TGF-α mRNA. Serum-free medium conditioned by T 3 M 4 and ASPC-1 cells contained significant amounts of TGF-α protein. Although unlabeled TGF-α readily competed with 125 I-labeled EGF for binding, each cell line exhibited lower surface binding and internalization of 125 I-labeled TGF-α as compared to 125 I-labeled EGF. Both TGF-α and EGF significantly enhanced the anchorage-independent growth of PANC-1, T 3 M 4 , and ASPC-1 cells. However, TGF-α was 10- to 100-fold more potent than EGF. These findings suggest that the concomitant overexpression of EGF receptors and production of TGF-α may represent an efficient mechanism for certain cancer cells to obtain a growth advantage

  13. CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

    Directory of Open Access Journals (Sweden)

    Capra Valérie

    2006-03-01

    Full Text Available Abstract Background Cysteine-containing leukotrienes (cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC proliferation. We used human ASMC (HASMC to identify the signal transduction pathway(s of the leukotriene D4 (LTD4-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS was estimated by measuring dichlorodihydrofluorescein (DCF oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX and phosphoinositide 3-kinase (PI3K inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF

  14. Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

    Science.gov (United States)

    Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

    2008-04-01

    Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplas