WorldWideScience

Sample records for human growth factors

  1. Epidermal growth factor (urogastrone) in human tissues.

    Science.gov (United States)

    Hirata, Y; Orth, D N

    1979-04-01

    Human epidermal growth factor (hEGF), which stimulates the growth of a variety of tissues, was first isolated from mouse submandibular glands, but is also excreted in large amounts (about 50 micrograms/day) in human urine and is probably identical to human beta-urogastrone (hUG), a potent inhibitor of stimulated gastric acid secretion. However, the primary tissue source of hEGF/hUG is as yet unknown. The hEGF/hUG in homogenates of human salivary glands and a wide variety of other endocrine and nonendocrine tissues was extracted by Amberlite CG-50 cation exchange chromatography and immune affinity chromatography using the immunoglobulin fraction of rabbit anti-hEGF serum covalently bound to agarose. The extracts were subjected to homologous hEGF RIA. Immunoreactive hEGF was found in extracts of adult submandibular gland, thyroid gland, duodenum, jejunum, and kidney, but not in several fetal tissues. The tissue immunoreactive hEGF was similar to standard hEGF in terms of immunoreactivity and elution from Sephadex G-50 Fine resin, but its concentrations were very low (1.3-5.5 ng/g wet tissue). Thus, it is not certain that these tissues represent the only source of the large amounts of hEGF/hUG that appear to be filtered by the kidneys each day.

  2. Human epidermal growth factor and the proliferation of human fibroblasts.

    Science.gov (United States)

    Carpenter, G; Cohen, S

    1976-06-01

    The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of 3H-thymidine incorporation after 20-24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.

  3. Pattern of hormone receptors and human epidermal growth factor ...

    African Journals Online (AJOL)

    2015-02-05

    Feb 5, 2015 ... Key words: Breast cancer, human epidermal growth factor receptor 2/neu, immunohistochemistry, ... therapy.[6‑8] Of all these prognostic and predictive factors, ... one of the biggest private medical laboratories in Nigeria.

  4. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  5. Immunolocalization of transforming growth factor alpha in normal human tissues

    DEFF Research Database (Denmark)

    Christensen, M E; Poulsen, Steen Seier

    1996-01-01

    the distribution of the growth factor in a broad spectrum of normal human tissues. Indirect immunoenzymatic staining methods were used. The polypeptide was detected with a polyclonal as well as a monoclonal antibody. The polyclonal and monoclonal antibodies demonstrated almost identical immunoreactivity. TGF...

  6. Basic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor-1in Human Meningiomas

    Institute of Scientific and Technical Information of China (English)

    YI Wei; CHEN Jian; Filimon H. Golwa; XUE Delin

    2005-01-01

    The expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in human meningiomas and the relationships between their expression and the tumors' histological features and angiogenesis were investigated by means of immunohistochemical technique. The expression of bFGF and FGFR-1 was detected by antibody of bFGF or FGFR-1.The tumors' angiogenesis was evaluated by microvascular density (MVD) and, which was observed by use of CD34-antibody immunohistochemically. The results showed that there were varied degrees of the expression of bFGF and FGFR-1 proteins in meningiomas. The expression was correlated with the tumors' histological characters and angiogenesis. It was concluded that bFGF and FGFR-1 might play important roles in meningiomas' angiogenesis and proliferation. The expression positive rate of bFGF and FGFR-1 may provide an indication of evaluating the histological and malignant degree of the tumor.

  7. Transforming growth factor (TGF)-. alpha. in human milk

    Energy Technology Data Exchange (ETDEWEB)

    Okada, Masaki; Wakai, Kae; Shizume, Kazuo (Research Institute for Growth Sciences, Tokyo (Japan)); Iwashita, Mitsutoshi (Tokyo Women' s Medical College (Japan)); Ohmura, Eiji; Kamiya, Yoshinobu; Murakami, Hitomi; Onoda, Noritaka; Tsushima, Toshio

    1991-01-01

    Transforming growth factor (TGF)-{alpha} and epidermal growth factor (EGF) were measured in human milk by means of homologous radioimmunoassay. As previously reported, EGF concentration in the colostrum was approximately 200 ng/ml and decreased to 50 ng/ml by day 7 postpartum. The value of immunoreactive (IR)-TGF-{alpha} was 2.2-7.2 ng/ml, much lower than that of EGF. In contrast to EGF, the concentration of IR-TGF-{alpha} was fairly stable during the 7 postpartum days. There was no relationship between the concentrations of IR-TGF-{alpha} and IR-EGF, suggesting that the regulatory mechanism in the release of the two growth factors is different. On gel-chromatography using a Sephadex G-50 column, IR-EGF appeared in the fraction corresponding to that of authentic human EGF, while 70%-80% of the IR-TGF-{alpha} was eluted as a species with a molecular weight greater than that of authentic human TGF-{alpha}. Although the physiological role of TGF-{alpha} in milk is not known, it is possible that it is involved in the development of the mammary gland and/or the growth of newborn infants.

  8. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development.

    Science.gov (United States)

    Janér, Joakim; Andersson, Sture; Haglund, Caj; Karikoski, Riitta; Lassus, Patrik

    2008-08-01

    We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of 10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression in capillary endothelium in bronchopulmonary dysplasia

  9. Purification of human platelet-derived growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Raines, E.W.; Ross, R.

    1985-01-01

    The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. (/sup 3/H)thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay.

  10. Vancouver Experience of Recombinant Human Platelet-Derived Growth Factor.

    Science.gov (United States)

    Younger, Alistair; Penner, Murray; Montijo, Harvey E

    2016-12-01

    Joint arthrodesis utilizing autogenous bone graft remains the gold standard of treatment in fusion procedures of the foot and ankle. Graft harvest, however, has been associated with increased morbidity to patients as well as increased costs. With this in mind, multiple clinical studies have evaluated the efficacy of recombinant human platelet-derived growth factor (rh-PDGF-BB) with beta-tricalcium phosphate (B-TCP) to augment in foot and ankle arthrodesis with favorable results. These factors have led to the increased use of rh-PDGF-BB with B-TCP in Vancouver with good clinical results. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Connective Tissue Growth Factor Expression in Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Amrita DOSANJH

    2006-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor β and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of α type I collagen, 5α-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody.The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.

  12. Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF.

    Directory of Open Access Journals (Sweden)

    Yonghua He

    Full Text Available Necrotizing enterocolitis (NEC is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF, typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

  13. EXPRESSION OF EPIDERMAL GROWTH FACTOR, TRANSFORMING GROWTH FACTOR-a AND THEIR RECEPTOR IN HUMAN PITUITARY TUMORS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Long

    2001-01-01

    [1]LIU Xu-wen, FU Pei-yu, GAO Zhi-xian. Expression of epidermal growth factor receptors in human glioma [J]. Chin J Neurosurgery 1998; 14:71.[2]Wong AJ, Ruppert JM, Bigner SH, et al. Structural alterations of the epidermal growth factor receptor gene in human gliomas [J]. Proc Natl Acad Sci USA 1992; 89:4309.[3]Webster J, Ham J, Bevan JS. Preliminary characterization of growth factors secreted by human pituitary tumors [J]. J Clin Endocrinol Metab 1991; 72:687.[4]Klibanski A. Nonsecreting pituitary tumors [J]. Endocrinol Metab Clin North Am 1987; 16:793.[5]LeRiche VK, Asa SL, Ezzat S. Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness [J]. J Clin Endocrinol Metab 1996; 81:656.

  14. Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor.

    OpenAIRE

    Francki, A.; Uciechowski, P.; Floege, J; von der Ohe, J.; Resch, K.; Radeke, H. H.

    1995-01-01

    For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine growth-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic fibroblast growth factor (bFGF). The ba...

  15. 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....../inactive endometria and seven of 13 (54%) endometria with adenomatous hyperplasia were EGF-R positive, with an immunostaining pattern rather similar to that of the carcinomas....

  16. Expression of epidermal growth factor receptors in human brain tumors.

    Science.gov (United States)

    Libermann, T A; Razon, N; Bartal, A D; Yarden, Y; Schlessinger, J; Soreq, H

    1984-02-01

    The expression of receptors for epidermal growth factor (EGF-R) was determined in 29 samples of brain tumors from 22 patients. Primary gliogenous tumors, of various degrees of cancer, five meningiomas, and two neuroblastomas were examined. Tissue samples were frozen in liquid nitrogen immediately after the operation and stored at -70 degrees until use. Cerebral tissue samples from 11 patients who died from diseases not related to the central nervous system served as controls. Immunoprecipitation of functional EGF-R-kinase complexes revealed high levels of EGF-R in all of the brain tumors of nonneuronal origin that were examined. The level of EGF-R varied between tumors from different patients and also between specimens prelevated from different areas of the same tumor. In contrast, the levels of EGF-R from control specimens were invariably low. The biochemical properties of EGF-R in brain tumor specimens were found to be indistinguishable from those of the well-characterized EGF-R from the A-431 cell line, derived from human epidermoid carcinomas. Human brain EGF-R displays a molecular weight of 170,000 by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. It is phosphorylated mainly in tyrosine residues and shows a 2-dimensional phosphopeptide map similar to that obtained with the phosphorylated EGF-R from membranes of A-431 cells. Our observations suggest that induction of EGF-R expression may accompany the malignant transformation of human brain cells of nonneuronal origin.

  17. Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent

    NARCIS (Netherlands)

    Gosens, Reinout; Schaafsma, D.; Grootte Bromhaar, M.M; Vrugt, B.; Zaagsma, Hans; Meurs, Herman; Nelemans, Herman

    2004-01-01

    Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-06-01

    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) ..mu..g/total vol, or 29.7 +- 1.1 and 39.8 +- 1.7 ..mu..g/g creatinine, respectively. Excretion by females taking oral contraceptives was significantly greater (60.1 +- 2.7 ..mu..g/g creatinine; P < 0.01) than that by females who were not. Recent evidence suggests the probable identity of hEGF and ..beta..-urogastrone, a potent inhibitor of gastric acid secretion. Adult males with active peptic ulcer disease appeared to have lower urinary RIA-hEGF excretion (22.9 +- 2.6 ..mu..g/g creatinine) than normal men, but this was not significant (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.

  19. Mecasermin (recombinant human insulin-like growth factor I).

    Science.gov (United States)

    Rosenbloom, Arlan L

    2009-01-01

    Growth hormone (GH) exercises its growth effects by stimulating insulin-like growth factor I (IGF-I) synthesis in the liver (endocrine IGF-I) and by inducing chondrocyte differentiation/replication and local production of IGF-I (paracrine/autocrine IGF-I). Injectable recombinant human (rh)IGF-I (mecasermin) has been available for nearly 20 years for treatment of the rare instances of GH insensitivity caused by GH receptor defects or GH-inhibiting antibodies. Full restoration of normal growth, as occurs with rhGH replacement of GH deficiency, is not seen, presumably because only the endocrine deficiency is addressed. RhIGF-I has also been effective as an insulin-sensitizing agent in severe insulin-resistant conditions. Although the insulin-sensitizing effect may benefit both type 1 and type 2 diabetes, there are no ongoing clinical trials because of concern about risk of retinopathy and other complications. Promotion of rhIGF-I for treatment of idiopathic short stature has been intensive, with neither data nor rationale suggesting that there might be a better response than has been documented with rhGH. Other applications that have either been considered or are undergoing clinical trial are based on the ubiquitous tissue-building properties of IGF-I and include chronic liver disease, cystic fibrosis, wound healing, AIDS muscle wasting, burns, osteoporosis, Crohn's disease, anorexia nervosa, Werner syndrome, X-linked severe combined immunodeficiency, Alzheimer's disease, muscular dystrophy, amyotrophic lateral sclerosis, hearing loss prevention, spinal cord injury, cardiovascular protection, and prevention of retinopathy of prematurity. The most frequent side effect is hypoglycemia, which is readily controlled by administration with meals. Other common adverse effects involve hyperplasia of lymphoid tissue, which may require tonsillectomy/adenoidectomy, accumulation of body fat, and coarsening of facies. The anti-apoptotic properties of IGF-I are implicated in cancer

  20. Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-hua; WEI Wei; XU Hao; WANG Yan-yan; WU Wen-xi

    2007-01-01

    Background Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.Methods Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA.The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.Results Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.Conclusion This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

  1. Topography of human placental receptors for epidermal growth factor.

    Science.gov (United States)

    Rao, C V; Ramani, N; Chegini, N; Stadig, B K; Carman, F R; Woost, P G; Schultz, G S; Cook, C L

    1985-02-10

    These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

  2. Maternal serum placental growth hormone, but not human placental lactogen or insulin growth factor-1, is positively associated with fetal growth in the first half of pregnancy

    DEFF Research Database (Denmark)

    Pedersen, N G; Juul, A; Christiansen, M

    2010-01-01

    To investigate if maternal levels of human placental lactogen (hPL), placental growth hormone (PGH) and insulin-like growth factor-1 (IGF-1) are associated with growth rate of the biparietal diameter (BPD) in the first half of pregnancy.......To investigate if maternal levels of human placental lactogen (hPL), placental growth hormone (PGH) and insulin-like growth factor-1 (IGF-1) are associated with growth rate of the biparietal diameter (BPD) in the first half of pregnancy....

  3. Effect of transforming growth factor-β1 on human intrahepatic cholangiocarcinoma cell growth

    Institute of Scientific and Technical Information of China (English)

    Tetsuya Shimizu; Takashi Tajiri; Shigeki Yokomuro; Yoshiaki Mizuguchi; Yutaka Kawahigashi; Yasuo Arima; Nobuhiko Taniai; Yasuhiro Mamada; Hiroshi Yoshida; Koho Akimaru

    2006-01-01

    AIM: To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on intrahepatic cholangiocarcinoma (ICC).METHODS: We investigated the effects of TGF-β1 on human ICC cell lines (HuCCT1, MEC, and HuH-28) by monitoring the influence of TGF-β1 on tumor growth and interleukin-6 (IL-6) expression in ICC cells.RESULTS: All three human ICC cell lines produced TGF-β1 and demonstrated accelerated growth in the presence of TGF-β1 with no apoptotic effect. Studies on HuCCT1 revealed a TGF-β1-induced stimulation of the expression of TGF-β1, as well as a decrease in TGF-β1 mRNA expression induced by neutralizing anti-TGF-β1 antibody. These results indicate that TGF-β1 stimulates the production and function of TGF-β1 in an autocrine fashion. Further, IL-6 secretion was observed in all three cell lines and exhibited an inhibitory response to neutralizing anti-TGF-β1 antibody. Experiments using HuCCT1 revealed a TGF-β1-induced acceleration of IL-6 protein expression and mRNA levels. These findings demonstrate a functional interaction between TGF-β1 and IL-6. All three cell lines proliferated in the presence of IL-6. In contrast, TGF-β1 induced no growth effect in HuCCT1 in the presence of small interfering RNA against a specific cell surface receptor of IL-6 and signal transducer and activator of transcription-3.CONCLUSION: ICC cells produce TGF-β1 and confer a TGF-β1-induced growth effect in an autocrine fashion.TGF-β1 activates IL-6 production, and the functional interaction between TGF-β1 and IL-6 contributes to ICC cell growth by TGF-β1.

  4. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Jun [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Xiang, Jun-Jian, E-mail: txjj@jnu.edu.cn [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China); Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Li, Dan [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Deng, Ning; Wang, Hong; Gong, Yi-Ping [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China)

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  5. EXPRESSION OF GROWTH-FACTORS AND GROWTH-FACTOR RECEPTORS IN NORMAL AND TUMOROUS HUMAN THYROID TISSUES

    NARCIS (Netherlands)

    van der Laan, B.F.A.M.; FREEMAN, JL; ASA, SL

    1995-01-01

    A number of growth factors have been implicated as stimuli of thyroid cell proliferation; overexpression of these growth factors and/or their receptors may play a role in the growth of thyroid tumors. To determine if immunohistochemical detection of growth factors and/or their receptors correlates w

  6. P01.02FIBROBLAST GROWTH FACTOR 4 CONTRIBUTES TO 3-DIMENSIONAL GROWTH OF HUMAN GLIOBLASTOMA

    Science.gov (United States)

    Lötsch, D.; Englinger, B.; Pichler, J.; Hainfellner, J.; Marosi, C.; Czech, T.; Knosp, E.; Buchroithner, J.; Spiegl-Kreinecker, S.; Berger, W.

    2014-01-01

    Glioblastoma growth is driven by receptor tyrosine kinase (RTK)-mediated signals. One of the RTK systems recently coming into focus are the fibroblast growth factor (FGF) high-affinity receptors (FGFR1-FGFR4) due to mutation, overexpression or translocation in several cancer types. FGF/FGFR represents a complex signal network with essential functions in embryonic development, tissue homeostasis and wound healing but also for malignant transformation and growth as well as tumor neoangiogenesis and therapy failure. Several studies have suggested a role of FGFRs in human glioblastoma whereby the information on FGFR4 is sparse. Here we investigated whether FGFR4 as compared to FGFR1 blockade impacts on glioblastoma growth in vitro and in vivo. Both in human glioblastoma cell lines (N = 8) and primary cell cultures from clinical samples (N = 26) we found a widespread expression of several FGFs (e.g. FGF1, FGF2, and FGF5) but also a significant overexpression of FGFR1 and FGFR4 in distinct subgroups as compared to non-malignant brain primo cell cultures. Regarding FGFR1 mRNA, all glioma cell models investigated expressed in addition to the FGFR1-IIIb also the mesenchymal and more oncogenic FGFR1-IIIc splice variant. Application of the FGFR inhibitors (nintedanib, ponatinib) as well as expression of dominant-negative (dn) versions of FGFR1 and FGFR4 significantly reduced in vitro cell growth and clonogenicity in the tested glioma cell models whereby dnFGFR1 tended to be more efficient than dnFGFR4. Accordingly, both dominant-negative FGFRs induced significant apoptosis whereby the effects of dnFGFR1 were again significantly stronger. Surprisingly, the inhibitory effects on anchorage-independent growth in soft agar were opposite with significant mitigation by dnFGFR1 but almost complete blockade by dnFGFR4 in the majority of the glioblastoma models analysed. Additionally, neurosphere formation, indicative for the presence of glioma stem cells, was profoundly reduced by

  7. Insulin infusion reduces hepatocyte growth factor in lean humans

    DEFF Research Database (Denmark)

    de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia;

    2013-01-01

    OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty......-two participants (10 women/12 men, BMI 20.6-34.5 kg/m(2), age 18-49 years) underwent a hyperinsulinemic euglycemic clamp with measurement of HGF at baseline and steady state. Relationships between baseline HGF, anthropometrics, triglycerides, liver enzymes, c-reactive protein and adiponectin were also evaluated...

  8. Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

    Science.gov (United States)

    Broughton, Marianne Nordlund; Westgaard, Arne; Paus, Elisabeth; Øijordsbakken, Miriam; Henanger, Karoline J; Naume, Bjørn; Bjøro, Trine

    2017-06-01

    The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, human epidermal growth factor receptor 3, human epidermal growth factor receptor 4, human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with

  9. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  10. EXPRESSION OF EPIDERMAL GROWTH FACTOR, TRANSFORMING GROWTH FACTOR-a AND THEIR RECEPTOR IN HUMAN PITUITARY TUMORS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To explore the role of growth factor autocrine stimulation in the pathogenesis of human pituitary tumors. Methods: The expression of EGF, TGF-a and EGFR were studied by immunohisto-chemical method on paraffin-embedded sections of 30 cases pituitary tumor. Results: EGFR and its ligands EGF, TGF-a expressed in majority of pituitary tumors. The expression of EGFR and its ligands varied with cells' intensity, density and type. Conclusion: The EGF autocrine stimulating exerted in the pituitary tumor development process, that tyrosine kinases inhibitors may be useful for pituitary tumors treatment.

  11. Estrogens and Insulin-Like Growth Factor 1 Modulate Neoplastic Cell Growth in Human Cholangiocarcinoma

    Science.gov (United States)

    Alvaro, Domenico; Barbaro, Barbara; Franchitto, Antonio; Onori, Paolo; Glaser, Shannon S.; Alpini, Gianfranco; Francis, Heather; Marucci, Luca; Sterpetti, Paola; Ginanni-Corradini, Stefano; Onetti Muda, Andrea; Dostal, David E.; De Santis, Adriano; Attili, Adolfo F.; Benedetti, Antonio; Gaudio, Eugenio

    2006-01-01

    We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-α, ER-β, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-α was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-β, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17β-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17β-estradiol and IGF-1 were associated with enhanced protein expression of ER-α, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-β. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma. PMID:16936263

  12. Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

    Institute of Scientific and Technical Information of China (English)

    HAI-TAO WANG; BIN WANG; ZHI-JUN LIU; ZHI-QIANG BAI; LING LI; HAI-YAN LIU; DONG-MENG QIAN; ZHI-YONG YAN; XU-XIA SONG

    2009-01-01

    Objectives To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P<0.05). Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

  13. HCG variants, the growth factors which drive human malignancies

    Science.gov (United States)

    Cole, Laurence A

    2012-01-01

    The term human chorionic gonadotropin (hCG) refers to a group of 5 molecules, each sharing the common amino acid sequence but each differing in meric structure and carbohydrate side chain structure. The 5 molecules are each produced by separate cells and each having separate biological functions. hCG and sulfated hCG are hormones produced by placental syncytiotrophoblast cells and pituitary gonadotrope cells. Hyperglycosylated hCG is an autocrine produced by placental cytotrophoblast cells. Hyperglycosylated hCG drives malignancy in placental cancers, and in testicular and ovarian germ cell malignancies. hCGβ and hyperglycosylated hCGβ are autocrines produce by most advanced malignancies. These molecules, particularly the malignancy promoters are presented in this review on hCG and cancer. hCGβ and hyperglycosylated hCGβ are critical to the growth and invasion, or malignancy of most advanced cancers. In many ways, while hCG may appear like a nothing, a hormone associated with pregnancy, it is not, and may be at the center of cancer research. PMID:22206043

  14. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    OpenAIRE

    2011-01-01

    International audience; This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarifi...

  15. SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia

    Science.gov (United States)

    Musa, Hassan; Kline, Crystal F.; Sturm, Amy C.; Murphy, Nathaniel; Adelman, Sara; Wang, Chaojian; Yan, Haidun; Johnson, Benjamin L.; Csepe, Thomas A.; Kilic, Ahmet; Higgins, Robert S. D.; Janssen, Paul M. L.; Fedorov, Vadim V.; Weiss, Raul; Salazar, Christina; Hund, Thomas J.; Pitt, Geoffrey S.; Mohler, Peter J.

    2015-01-01

    Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins. PMID:26392562

  16. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor)

    Energy Technology Data Exchange (ETDEWEB)

    Winkles, J.A.; Friesel, R.; Burgess, W.H.; Howk, R.; Mehlman, T.; Weinstein, R.; Maciag, T.

    1987-10-01

    The control of vascular endothelial and muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical cells also synthesize an HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with /sup 125/I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.

  17. P01.02FIBROBLAST GROWTH FACTOR 4 CONTRIBUTES TO 3-DIMENSIONAL GROWTH OF HUMAN GLIOBLASTOMA

    OpenAIRE

    Lötsch, D.; Englinger, B.; Pichler, J; Hainfellner, J; Marosi, C; Czech, T.; Knosp, E.; Buchroithner, J; Spiegl-Kreinecker, S.; Berger, W

    2014-01-01

    Glioblastoma growth is driven by receptor tyrosine kinase (RTK)-mediated signals. One of the RTK systems recently coming into focus are the fibroblast growth factor (FGF) high-affinity receptors (FGFR1-FGFR4) due to mutation, overexpression or translocation in several cancer types. FGF/FGFR represents a complex signal network with essential functions in embryonic development, tissue homeostasis and wound healing but also for malignant transformation and growth as well as tumor neoangiogenesis...

  18. Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

    Directory of Open Access Journals (Sweden)

    Poyil Pratheeshkumar

    Full Text Available Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

  19. Nerve growth factor in human semen: Effect of nerve growth factor on the normozoospermic men during cryopreservation process

    Science.gov (United States)

    Saeednia, Sara; Bahadoran, Hosein; Amidi, Fardin; Asadi, Mohammad Hosein; Naji, Mohammad; Fallahi, Parvin; Nejad, Nahid Ataie

    2015-01-01

    Objective(s): Although routinely applied in assisted reproductive technology, human sperm cryopreservation is not a completely successful procedure. Adverse effects of cryopreservation on the fertilization capacity, motility, morphology, and viability of spermatozoa have been proven; cryopreservation has also shown a role in sperm DNA fragmentation and infertility. The post-thaw survival of spermatozoa improved after addition of supplementation of antioxidant molecules to freezing media. Nerve growth factor (NGF) as one of the prosurvival substances has gained great attention in recent years. The aim of this study was the usage of NGF as prosurvival factor after cryopreservation process of human semen samples to assess the motility and viability of sperm, nitric oxide (NO) concentration, and DNA fragmentation in normozoospermic men. Materials and Methods: Semen samples were collected from 25 normozoospermic men and were divided into fresh semen samples as control group, frozen–thawed semen samples without addition of exogenous NGF, and three groups of semen samples cryopreserved with addition of exogenous NGF (0.5, 1, and 5 ng/ml) in freezing medium. Viability was assessed by eosin-negrosin staining technique. Motility was evaluated with inverted microscope. NO concentration and apoptosis content were measured with flow cytometry. Results: Results showed that exogenous NGF at 0.5 ng/ml could significantly (P-value <0.05) influence viability, motility, nitric oxide, and DNA fragmentation content. Conclusion: Exogenous NGF as cryoprotectant improved sperm viability and motility, increased intracellular NO concentration, and decreased apoptosis content in normal human spermatozoa. PMID:25945243

  20. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  1. Molecular cloning of a human gene that is a member of the nerve growth factor family

    Energy Technology Data Exchange (ETDEWEB)

    Jones, K.R.; Reichardt, L.F. (Howard Hughes Medical Institute, San Francisco, CA (USA))

    1990-10-01

    Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, share substantial amino acid sequence identity. The authors have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene, neurotrophin-3, that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development.

  2. Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

    Institute of Scientific and Technical Information of China (English)

    Ming Xiu; Ya-Hui Liu; David R Brigstock; Fang-Hui He; Rui-Juan Zhang; Run-Ping Gao

    2012-01-01

    AIM:To determine the expression characteristics of connective tissue growth factor (CTGF/CCN2) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1 (TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1 (FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci (12.36 ± 6.08 vs 6.42 ± 2.35)or 9.4-fold in the surrounding stroma (60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2 (100 ng/mL) resulted in more of the cells transitioning into S phase (23.85 ± 2.35vs 10.94 ± 0.23),and induced a significant migratory (4.0-fold) and invasive (5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2

  3. Hepatocyte Growth Factor Suppresses Transforming Growth Factor-Beta-1 and Type III Collagen in Human Primary Renal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Shan Mou

    2009-11-01

    Full Text Available Tubulointerstitial changes in the diabetic kidney correlate closely with renal fibrosis, and transforming growth factor-beta-1 (TGF-β1 is thought to play a key role in this process. In contrast, hepatocyte growth factor (HGF has shown therapeutic effects on injured renal tubules in animal models. This study was undertaken to test the hypothesis that the preventive effects of HGF may result from interventions in TGF-β1-mediated signaling and collagen III secretion. We examined the expression of HGF/HGF receptor (c-Met and TGF-β1 in renal fibroblasts at multiple time points. The effects of recombinant human HGF on TGF-β1 expression were studied by RT-PCR and Western blotting, and the levels of collagen III were measured by ELISA. In the high-glucose condition, the expression of HGF and c-Met in renal fibroblasts was detected as early as 6 hours following cell culture while the level of TGF-β1 peaked at 96 hours. The addition of recombinant human HGF to the culture media dose-dependently inhibited TGF-β1 mRNA expression and reduced collagen III secretion by 34%. These results indicate that, during hyperglycemia, HGF inhibits TGF-β1 signaling and type III collagen activation in interstitial fibroblasts. Furthermore, we should recognize that changes in the balance between HGF and TGF-β1 might be decisive in the pathogenesis of chronic renal fibrosis. Therefore, administration of HGF to restore this balance may offer a novel therapeutic intervention in managing renal fibrogenesis in diabetic nephropathy.

  4. The human insulin-like growth factor II gene contains two development-specific promoters

    NARCIS (Netherlands)

    Pagter-Holthuizen, P. de; Jansen, M.; Schaik, F.M.A.; Kammen, R. van der; Oosterwijk, C.; Brande, J.L. van den; Sussenbach, J.S.

    1987-01-01

    The insulin-like growth factors (IGF) play an important role in fetal and postnatal development. Recently, the nucleotide sequences of the cDNAs encoding IGF-I and IGF-II and part of the human IGF genes were reported. In this communication we describe two distinct IGF-II cDNAs isolated from a human

  5. Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

    OpenAIRE

    Hadadian, Shahin; Shamassebi, Dariush Norouzian; Mirzahoseini, Hasan; Shokrgozar, Mohamad Ali; Bouzari, Saeid; Sepahi, Mina

    2015-01-01

    Background: Human basic fibroblast growth factor (hBFGF) is a heparin-binding growth factor and stimulates the proliferation of a wide variety of cells and tissues causing survival properties and its stability and biological activity improvements have received much attention. Materials and Methods: In the present work, hBFGF produced by engineered Escherichia coli and purified by cation exchange and heparin affinity chromatography, was PEGylated under appropriate condition employing 10 kD pol...

  6. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Mona Alibolandi

    2011-01-01

    Full Text Available This work describes the integration of expanded bed adsorption (EBA and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

  7. Expression of human acidic fibroblast growth factor in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    YU Ying; CAI Shaoxi; Harald G. WERIRICH; XIA Yuxian

    2003-01-01

    Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoriS KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mg.L-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 1 7 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.

  8. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F;

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insuli...

  9. Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation.

    Science.gov (United States)

    Kasper, S; Friesen, H G

    1986-01-01

    We report the secretion from human pituitary tumor fragments in organ culture of a potent mitogen for chondrocyte proliferation. Primary human pituitary cell and organ cultures were established from pituitary fragments obtained from patients with acromegaly, prolactinomas, and nonfunctional adenomas. The conditioned culture medium contained a mitogenic factor(s) that stimulated rabbit fetal chondrocyte proliferation, causing up to an 8-fold increase in cell number when added to Ham's F-10 medium in the presence of 10% fetal bovine serum. Blood leaking into the surgical field after the adenomectomy is known to contain very high concentrations of pituitary hormones. Serum samples, obtained from this venous "ooze" collected at the site of pituitary surgery, also were found to contain chondrocyte growth-promoting activity. Some venous serum samples stimulated chondrocyte proliferation in a dose-dependent manner down to a 1:10 dilution of 1 microliter serum, indicating that the material being secreted was very potent indeed. Gel filtration on Sephadex G-100 and analytical gel isoelectric focusing of culture media or serum samples from the pituitary fossa demonstrated that the growth factor secreted from the pituitary tumor fragments as well as from the venous serum is similar, if not identical, to chondrocyte growth factor (mol wt, 43,000; pI 7.6-7.9) purified from human pituitaries collected at autopsy. These results suggest that the chondrocyte growth-promoting factor(s) may not only be secreted by pituitary tumor fragments but by normal human pituitary tissue as well.

  10. Cloning, Expression and Functional Characterization of In-House Prepared Human Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Hassan Rassouli

    2013-01-01

    Full Text Available Objective: Human basic fibroblast growth factor (bFGF plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs.Materials and Methods: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability.Results: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo.Conclusion: The results show no significant difference in function between our in-house produced and commercialized bFGF.

  11. Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells*

    Institute of Scientific and Technical Information of China (English)

    Yan Zhang; Junmei Zhou; Zhenfu Fang; Manxi Jiang; Xuejin Chen

    2013-01-01

    The difference between Noggin and basic fibroblast growth factor for the neural precursor differen-tiation from human embryonic stem cel s has not been studied. In this study, 100 µg/L Noggin or 20 µg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen-tiate human embryonic stem cel s H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro-scope. Immunofluorescence staining revealed expression levels of Nestin,β-III Tubulin and Sox-1 were higher in the induced cel s and reverse-transcription PCR showed induced cel s expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cel differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in-creases the differentiation of neural precursors.

  12. Immunocytochemical localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 of the human deciduous molar tooth germ development in the human fetus.

    Science.gov (United States)

    Miwa, Yoko; Fujita, Toshiya; Sunohara, Masataka; Sato, Iwao

    2008-01-01

    Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel endothelial development. We used immunohistochemical methods to demonstrate the localization of VEGF and its receptors, showing the specific expression pattern of VEGF and VEGF receptor in the human deciduous tooth from the cap to late bell stages in the human fetus. Immunoreactivity to VEGF and its receptor VEGF receptor-2 (VEGFR-2) was intensely positive in the inner enamel epithelium at the cap stage and ranged from negative to moderately positive in the bell stage. At the late bell stage, VEGF immunoreactivity was mainly positive but weak for VEGFR-2. The intensity of VEGF and VEGFR-2 in odontoblasts increases from cap stage to late bell stage. We postulate that the dissimilar expression of VEGF in inner enamel epithelium, ameloblast and odontoblast during each stage of human tooth development may affect tooth germ formation.

  13. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  14. Hexamethylenebisacetamide (HMBA) is a growth factor for human, ovine and porcine thyroid cells.

    Science.gov (United States)

    Fayet, G; Amphoux-Fazekas, T; Aouani, A; Hovsépian, S

    1996-03-01

    Hexamethylenebisacetamide (HMBA) provokes in murine erythroleukemia cells (MELC) a commitment to terminal differentiation leading to the activation of the expression of hemoglobin. HMBA has been tested also in other cells from colon cancer, melanoma or lung cancer. However it has not yet been tested in the thyroid. We demonstrate in this paper that HMBA in kinetics and concentration-response experiments increases the proliferation of human thyroid cells isolated from Graves'-Basedow patients. It also acts like a growth factor for ovine and porcine thyroid cells, respectively, from the OVNIS line and the ATHOS line. This molecule which is a differentiating factor in the MELC system and a growth factor in human thyroid cell cultures represents a potential to get human thyroid cell lines expressing specialized functions.

  15. A new method for high yield purification of type beta transforming growth factor from human platelets

    NARCIS (Netherlands)

    Eijnden-van Raaij, A.J.M. van den; Koornneef, I.; Zoelen, E.J.J. van

    1988-01-01

    A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer

  16. Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue

    DEFF Research Database (Denmark)

    Herchenhan, Andreas; Bayer, Monika L.; Eliasson, Pernilla

    2015-01-01

    OBJECTIVE: Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We...

  17. Response of growth and myogenic factors in human skeletal muscle to strength training

    NARCIS (Netherlands)

    Liu, Y.; Heinichen, M.; Wirth, K.; Schmidtbleicher, D.; Steinacker, J. M.

    2008-01-01

    Objective: To investigate the response to different strength training techniques of growth and myogenic factors in human skeletal muscle, with particular emphasis on satellite cell (SC) activation. Methods: 24 volunteers were divided into two groups and performed a 6-week strength training (group A

  18. Expression of the epidermal growth factor system in human middle ear cholesteatoma

    DEFF Research Database (Denmark)

    Thorup, Mette Bendixen; Munk, Mathias; Poulsen, Steen Seier

    2014-01-01

    the expression of the epidermal growth factor (EGF) system in human middle ear cholesteatoma. Methods: Forty-seven patients referred for surgery due to cholesteatoma were included in the study. Clinical data were collected. Biopsies of cholesteatoma and skin from the external ear canal were obtained during...

  19. RNA interference inhibits expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    CAI Chun-mei; SUN Bao-chen; LIU Xu-yang; WANG Jin-jin; LI Jun-fa; HAN Song; WANG Ning-li; LU Qing-jun

    2005-01-01

    @@ Choroidal neovascularization (CNV), a major cause of vision loss, is the result of the increased vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. It is important to inhibit the expression of VEGF protein in RPE cells.

  20. Human Vascular Endothelial Growth Factor cDNA Cloning and Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Human vascular endothelial growth factor (VEGF) cDNA was amplified by nested polymerase chain reaction method from the HL60 cells. Then a pCD-hVEGF165 recombinant plasmid was constructed. Rabbit osteoblasts were transfected with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer. The transient expressive results were detected by immunohistochemical method. It was observed that the expression of human VEGF gene was detected 72 h after transfecting distinctly.

  1. 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...... (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...

  2. Dauricine inhibits insulin-like growth factor-Ⅰ-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xu-dong TANG; Xin ZHOU; Ke-yuan ZHOU

    2009-01-01

    Aim: To investigate the effects of dauricine (Dau) on insulin-like growth factor-Ⅰ (IGF-Ⅰ)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).Methods: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-Ⅰ for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively.HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.Results: Dau significantly inhibited IGF-Ⅰ-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-Ⅰ.Mechanistically, Dau suppressed IGF-Ⅰ-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dan reduced IGF-Ⅰ-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-Ⅰ-induced invasion of HUVECs.Conclusion: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.

  3. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte Rønde;

    2004-01-01

    Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV-lipodystrophy....... These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin...... of bioactive IGF-I in HIV-lipodystrophy....

  4. Expression of Transforming Growth Factor-β in Cultured Normal Human Lens Epithelia Cells

    Institute of Scientific and Technical Information of China (English)

    黄渝侃; 魏厚仁

    2004-01-01

    Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RTPCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.

  5. Impact of Growth Factor Independence 1 in Human T-Cell Lymphomas

    DEFF Research Database (Denmark)

    Dabrowska, Magdalena Julia; Dybkær, Karen; Johansen, Preben

    2009-01-01

    Impact of Growth Factor Independence 1 in Human T-Cell Lymphomas; Pathogenic Potential Identified by Insertional Mutagenesis in a Murine T-Cell Lymphoma Model. Magdalena Julia Dabrowska *,1, Karen Dybkaer *,1, Preben Johansen *,2, Hans Erik Johnsen1 and Finn Skou Pedersen *,3 1 Department...... role in the development of MLV induced lymphomas and strongly indicates that retroviral insertional mutagenesis in murine models of human NHLs can be used to identify new genes involved in lymphomagenesis and, by use of functional assays, their impact on human lymphomas can be evaluated. Disclosures...

  6. Effect of placental factors on growth and function of the human fetal adrenal in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Riopel, L.; Branchaud, C.L.; Goodyer, C.G.; Zweig, M.; Lipowski, L.; Adkar, V.; Lefebvre, Y. (McGill Univ.-Montreal Children' s Hospital Research Institute, Quebec (Canada))

    1989-11-01

    Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: (1) maximal response to PM was 2-5 times greater; (2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; (3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.

  7. Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth.

    Science.gov (United States)

    Thornbrough, Joshua M; Hundley, Tom; Valdivia, Raphael; Worley, Micah J

    2012-01-01

    Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.

  8. A combined model of human erythropoiesis and granulopoiesis under growth factor and chemotherapy treatment.

    Science.gov (United States)

    Schirm, Sibylle; Engel, Christoph; Loeffler, Markus; Scholz, Markus

    2014-05-26

    Haematotoxicity of conventional chemotherapies often results in delays of treatment or reduction of chemotherapy dose. To ameliorate these side-effects, patients are routinely treated with blood transfusions or haematopoietic growth factors such as erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF). For the latter ones, pharmaceutical derivatives are available, which differ in absorption kinetics, pharmacokinetic and -dynamic properties. Due to the complex interaction of cytotoxic effects of chemotherapy and the stimulating effects of different growth factor derivatives, optimal treatment is a non-trivial task. In the past, we developed mathematical models of thrombopoiesis, granulopoiesis and erythropoiesis under chemotherapy and growth-factor applications which can be used to perform clinically relevant predictions regarding the feasibility of chemotherapy schedules and cytopenia prophylaxis with haematopoietic growth factors. However, interactions of lineages and growth-factors were ignored so far. To close this gap, we constructed a hybrid model of human granulopoiesis and erythropoiesis under conventional chemotherapy, G-CSF and EPO applications. This was achieved by combining our single lineage models of human erythropoiesis and granulopoiesis with a common stem cell model. G-CSF effects on erythropoiesis were also implemented. Pharmacodynamic models are based on ordinary differential equations describing proliferation and maturation of haematopoietic cells. The system is regulated by feedback loops partly mediated by endogenous and exogenous EPO and G-CSF. Chemotherapy is modelled by depletion of cells. Unknown model parameters were determined by fitting the model predictions to time series data of blood counts and cytokine profiles. Data were extracted from literature or received from cooperating clinical study groups. Our model explains dynamics of mature blood cells and cytokines after growth-factor applications in healthy volunteers

  9. Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing MCF-7 human breast cancer cells.

    Science.gov (United States)

    Ruohola, J K; Viitanen, T P; Valve, E M; Seppänen, J A; Loponen, N T; Keskitalo, J J; Lakkakorpi, P T; Härkönen, P L

    2001-05-15

    Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.

  10. Functional Development of the Human Gastrointestinal Tract: Hormone- and Growth Factor-Mediated Regulatory Mechanisms

    Directory of Open Access Journals (Sweden)

    Daniel Ménard

    2004-01-01

    Full Text Available The present review focuses on the control of gastrointestinal (GI tract development. The first section addresses the differences in general mechanisms of GI development in humans versus rodents, highlighting that morphogenesis of specific digestive organs and the differentiation of digestive epithelia occur not only at different stages of ontogeny but also at different rates. The second section provides an overview of studies from the author's laboratory at the Université de Sherbrooke pertaining to the development of the human fetal small intestine and colon. While both segments share similar morphological and functional characteristics, they are nevertheless modulated by distinct regulatory mechanisms. Using the organ culture approach, the author and colleagues were able to establish that hormones and growth factors, such as glucocorticoids, epidermal growth factor, insulin and keratinocyte growth factor, not only exert differential effects within these two segments, they can also trigger opposite responses in comparison with animal models. In the third section, emphasis is placed on the functional development of human fetal stomach and its various epithelial cell types; in particular, the glandular chief cells responsible for the synthesis and secretion of gastric enzymes such as pepsinogen-5 and gastric lipase. Bearing in mind that limitations of available cell models have, until now, greatly impeded the comprehension of molecular mechanisms regulating human gastric epithelial cell functions, the last section focuses on new human gastric epithelial cell models recently developed in the author's laboratory. These models comprise a novel primary culture system of human fetal gastric epithelium including, for the first time, functional chief cells, and human gastric epithelium cell lines cloned from the parental NCI-N87 strain. These new cells lines could serve important applications in the study of pathogenic action and epithelial

  11. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte R;

    2004-01-01

    Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV-lipodystrophy. The......Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV......-lipodystrophy. These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin...... study groups, including suppressed GH, and increased GHBP in LIPO, argue against GH resistance of GH-sensitive tissues in LIPO compared with NONLIPO; however, this notion awaits examination in dose-response studies. Furthermore, our data suggest that IGFBP-3 protease is a significant regulator...

  12. Mitogenic properties of insulin-like growth factors I and II, insulin-like growth factor binding protein-3 and epidermal growth factor on human breast stromal cells in primary culture.

    Science.gov (United States)

    Strange, Karen S; Wilkinson, Darcy; Edin, Glenn; Emerman, Joanne T

    2004-03-01

    Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal-epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.

  13. Improvement in skin wrinkles using a preparation containing human growth factors and hyaluronic acid serum.

    Science.gov (United States)

    Lee, Do Hyun; Oh, In Young; Koo, Kyo Tan; Suk, Jang Mi; Jung, Sang Wook; Park, Jin Oh; Kim, Beom Joon; Choi, Yoo Mi

    2015-02-01

    Skin aging is accompanied by wrinkle formation. At some sites, such as the periorbital skin, this is a relatively early phenomenon. We evaluated the anti-wrinkle effect of a preparation containing human growth factor and hyaluronic acid serum on periorbital wrinkles (crow's feet). In total, 23 Korean women (age range: 39-59 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. All the patients completed an 8-week trial of twice-daily application of human growth factor and hyaluronic acid serum on the entire face. Efficacy was based on a global photodamage score, photographs, and image analysis using replicas and visiometer analysis every 4 weeks. The standard wrinkle and roughness parameters used in assessing skin by visiometer were calculated and statistically analyzed. Periorbital wrinkles were significantly improved after treatment, with improvements noted both by physician's assessment and visiometer analysis. Topical application of human growth factor and hyaluronic acid was beneficial in reducing periorbital wrinkles.

  14. Oocyte maturation in humans: the role of gonadotropins and growth factors.

    Science.gov (United States)

    Gómez, E; Tarín, J J; Pellicer, A

    1993-07-01

    To determine the effect of FSH/LH in vivo and epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in vitro on human oocyte maturation. Oocyte-cumulus complexes were harvested from three different groups of patients: [1] unstimulated ovaries from women undergoing surgery; [2] multifollicular development achieved with a combination of FSH and LH in the absence of an ovulatory dose of hCG; and [3] oocyte-cumulus complexes retrieved after appropriate ovarian stimulation with FSH/LH and hCG for IVF purposes. In vitro fertilization program and patients undergoing surgery for benign disorders at the Instituto Valenciano de Infertilidad, Valencia, Spain. Oocyte-cumulus complexes from unstimulated ovaries collected at surgery by follicular puncture and washing. Oocyte-cumulus complexes from stimulated cycles obtained by ultrasound-guided transvaginal aspiration. Oocyte-cumulus complexes cultured in vitro in the absence or presence of different concentrations of EGF and IGF-I. Germinal vesicle breakdown and metaphase-II stage after 24 and 48 hours. Comparison of the spontaneous resumption of meiosis and metaphase II oocytes among groups showed significant differences between unstimulated and stimulated ovaries after 24 and 48 hours in culture. Administration of hCG accelerated the percentage of maturation by 24 hours. Further incubation of unstimulated oocyte-cumulus complexes with EGF and IGF-I significantly increased the percentage of metaphase-II oocytes after 24 and 48 hours in culture. Epidermal growth factor and IGF-I are able to augment spontaneous maturaion in immature human oocytes. Because spontaneous maturation is mainly observed when follicles have been exposed to pharmacological doses of hMG, it is suggested that increasing FSH levels within the follicle is coincident with the generation of a positive signal necessary to complete oocyte maturation in humans. This signal may be linked to the dynamics of growth factors within the follicle itself.

  15. CLONING AND SEQUENCING OF MATURED FRAGMENT OF HUMAN NEVER GROWTH FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    马巍; 吴玲; 王德利; 刘淼; 任惠民; 杨广笑; 王全颖

    2003-01-01

    Objective Molecular cloning and sequencing of the human matured fragment of human nerve growth factor(NGF) gene. Methods Extracting the human genomic DNA from the white blood cells as templates, the gene of NGF was cloned by using PCR and T-vector cloning method. Screening the positive clones and identified by the restriction enzymes, and then the cloned amplified fragment was sequenced and analyzed. Results DNA sequence comparison the cloned gene of NGF with the GenBank (V01511) sequence demonstrated that both of sequences were identical, 354bp length. Conclusion Cloning the NGF gene from the human genomic DNA has paved the way for further study on gene therapy of nerve system injury.

  16. Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation.

    Science.gov (United States)

    Zhang, Jinlong; Lian, Min; Cao, Peipei; Bao, Guofeng; Xu, Guanhua; Sun, Yuyu; Wang, Lingling; Chen, Jiajia; Wang, Yi; Feng, Guijuan; Cui, Zhiming

    2017-04-01

    Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs + bFGF + NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF + NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.

  17. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...... based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted...... antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors...

  18. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

  19. Reactivation of the insulin-like growth factor-Ⅱ signaling pathway in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Kai Breuhahn; Peter Schirmacher

    2008-01-01

    Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma (HCC). Especially the over-expression of the fetal growth factor IGF-Ⅱ, IGF-Ⅰ receptor (IGF-IR), and cytoplasmic downstream effectors such as insulin-receptor substrates (IRS) contribute to proliferation, anti-apoptosis, and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱ signaling during HCC development and progression. Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies, various experimental strategies have been developed, including neutralizing antibodies and selective receptor ki-nase inhibitors, with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

  20. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    Science.gov (United States)

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  1. Relationship between cyclooxygenase-2 and human epidermal growth factor receptor 2 in vascular endothelial growth factor C up-regulation and lymphangiogenesis in human breast cancer.

    Science.gov (United States)

    Bhattacharjee, Rabindra N; Timoshenko, Alexander V; Cai, Jing; Lala, Peeyush K

    2010-09-01

    Both cyclooxygenase (COX)-2 and human epidermal growth factor receptor (HER)-2 promote breast cancer progression; however, the relationship between the two molecules remains unclear. We utilized human breast cancer tissues and cell lines to examine whether COX-2 and HER-2 played independent or interdependent roles in vascular endothelial growth factor (VEGF)-C up-regulation and lymphangiogenesis. A paired correlation of immunodetectable levels of COX-2, VEGF-C, and HER-2 proteins and lymphovascular density (LVD; D2-40-immunolabeled) in 55 breast cancer specimens revealed a positive correlation between COX-2 and HER-2 irrespective of clinicopathological status. However COX-2 alone positively correlated with LVD. In 10 independent specimens, mRNA levels showed a positive correlation between HER-2 and COX-2 or VEGF-C but not LYVE-1 (lymphovascular endothelial marker). These findings implicate COX-2, but not HER-2, in breast cancer-associated lymphangiogenesis. Manipulation of the COX-2 or HER-2 genes in breast cancer cell lines varying widely in COX-2 and HER-2 expression revealed a direct role of COX-2 and an indirect COX-2 dependent role of HER-2 in VEGF-C up-regulation: (i) high VEGF-C expression in high COX-2/low HER-2 expressing MDA-MB-231 cells was reduced by siRNA-mediated down-regulation of COX-2, but not HER-2; (ii) integration of HER-2 in these cells simultaneously up-regulated COX-2 protein as well as VEGF-C secretion; and (iii) low VEGF-C secretion by high HER-2/low COX-2 expressing SK-BR-3 cells was stimulated by COX-2 overexpression. These findings of the primary role of COX-2 and the COX-2-dependent role of HER-2, if any, in VEGF-C up-regulation and lymphangiogenesis suggest that COX-2 inhibitors may abrogate lymphatic metastasis in breast cancer irrespective of HER-2 status. © 2010 Japanese Cancer Association.

  2. Signal peptide of eosinophil cationic protein upregulates transforming growth factor-alpha expression in human cells.

    Science.gov (United States)

    Chang, Hao-Teng; Kao, Yu-Lin; Wu, Chia-Mao; Fan, Tan-Chi; Lai, Yiu-Kay; Huang, Kai-Ling; Chang, Yuo-Sheng; Tsai, Jaw-Ji; Chang, Margaret Dah-Tsyr

    2007-04-01

    Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.

  3. Therapeutic angiogenesis induced by human hepatocyte growth factor (HGF) gene in rat myocardial ischemia models

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the feasibility of myocardial ischemia gene therapy, we cloned human hepatocyte growth factor gene from human placenta cDNA library by the RT-PCR method. Recombination adenovirus Ad-HGF was constructed by the method of co-transfection and homologous recombination of plasmids in 293 cells. Ad-HGF was amplified in 293 cells and purified through CsCl density gradient centrifugation. Ad-HGF could be expressed in rat primary myocardial cells and HGF secreted into the culture media, which was tested by ELISA. The distribution and persistence of adenovirus in rat were investigated by green fluorescence protein as a report gene. In vivo we found that intramyocardial administration of Ad-HGF could induce angiogenesis in rat myocardium after ligation of coronary artery. The results suggested that Ad-HGF was effective in vitro and in vivo, and the data for designing human trial of gene therapy-- mediated cardiac angiogenesis were provided.

  4. Development of a sensitive enzyme immunoassay for human epidermal growth factor (urogastrone).

    Science.gov (United States)

    Kurobe, M; Tokida, N; Furukawa, S; Ishikawa, E; Hayashi, K

    1986-04-15

    A sensitive two-site enzyme immunoassay (EIA) for human epidermal growth factor (hEGF) was developed, based on the sandwiching of an antigen between anti-hEGF IgG-coated polystyrene beads and anti-hEGF Fab'-linked peroxidase complex (horseradish peroxidase, EC. 1.11.1.7). This method has four advantages: the anti-hEGF Fab'-linked peroxidase complex is more stable than 125I-labelled antibody; the procedure is simple and rapid compared to bioassay; its discriminatory sensitivity is as low as 0.1 pg/assay tube; and serial dilution curves of unextracted human serum and urine samples all paralleled that of standard hEGF. The validity of the measurement of hEGF-like immunoreactivity in human serum and plasma is discussed.

  5. Expression, content, and localization of insulin-like growth factor I in human achilles tendon

    DEFF Research Database (Denmark)

    Olesen, Jens L; Heinemeier, Katja M; Langberg, Henning;

    2006-01-01

    by immunoflourometric assay, real-time PCR, and immunohistochemistry used to localize and determine expression of IGF-I and IGFBP-4 in 6 postmortem human Achilles tendons. Tendon tissue concentrations of IGF-I were found to be 0.53 +/- 0.10 ng/g. Furthermore, we demonstrated that IGF-I and IGFBP-4 are localized around......In animals insulin-like growth factor I (IGF-I) stimulates collagen production by fibroblasts and is expressed in tendons together with its binding protein 4 (IGFBP-4). However, the presence of IGF-I and IGFBP-4 in human tendon tissue is not described. Tissue IGF-I content was examined...... the tendon fibroblasts and that mRNA for IGF-I and IGFBP-4 can be determined in human tendon tissue. The present study adds support for the roles of IGF-I and IGFBP-4 in the regulation of tendon adaptive responses to mechanical loading....

  6. Basic Fibroblast Growth Factor-Mediated Overexpression of Vascular Endothelial Growth Factor in 1F6 Human Melanoma Cells is Regulated by Activation of PI-3K and p38 MAPK

    Directory of Open Access Journals (Sweden)

    Dennis Fontijn

    2009-01-01

    Full Text Available Background: 1F6 human melanoma xenografts overexpressing either the 18 kD (18kD form or all (ALL forms of human basic fibroblast growth factor (bFGF demonstrate an abundant number of microvessels and accelerated growth. We now examined whether bFGF mediates vascular endothelial growth factor (VEGF expression.

  7. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  8. Human brain tumor-associated urinary high molecular weight transforming growth factor: a high molecular weight form of epidermal growth factor.

    Science.gov (United States)

    Stromberg, K; Hudgins, W R; Dorman, L S; Henderson, L E; Sowder, R C; Sherrell, B J; Mount, C D; Orth, D N

    1987-02-15

    Urinary protein obtained from a patient with a highly malignant brain tumor (astrocytoma, grade IV) was adsorbed to trimethylsilyl controlled-pore glass beads and selectively eluted with acetonitrile to yield a high molecular weight (HMW) human transforming growth factor (hTGF). This HMW hTGF promoted clonogenic cell growth in soft agar and competed for membrane receptors with mouse epidermal growth factor. After surgical resection of the tumor, no HMW hTGF was found in urine. HMW hTGF generated a human EGF (hEGF) radioimmunoassay competitive binding curve similar to that of hEGF and parallel to that of a highly purified HMW form of hEGF previously reported to be present in trace concentrations in normal human urine. Both hEGF and HMW hEGF were clonogenic in soft agar, and their clonogenic activity as well as that of HMW hTGF was inhibited by anti-hEGF serum. Both HMW hTGF and HMW hEGF had 20 to 25% of the radioreceptor binding activity of hEGF. HMW hTGF purified from the pooled urine of several patients with malignant astrocytomas and HMW hEGF purified from normal control urine comigrated at Mr 33,000. Thus, HMW hTGF was indistinguishable from HMW hEGF in terms of apparent molecular size, epidermal growth factor receptor binding activity, epidermal growth factor immunoreactivity, and clonogenic activity. Urinary HMW hEGF/hTGF may be of tumor cell origin or may represent a response of normal host tissues to the tumor or its products.

  9. Intraarticular Sprifermin (Recombinant Human Fibroblast Growth Factor 18) in Knee Osteoarthritis

    DEFF Research Database (Denmark)

    Lohmander, L. S.; Hellot, S.; Dreher, D.

    2014-01-01

    Objective. To evaluate the efficacy and safety of intraarticular sprifermin (recombinant human fibroblast growth factor 18) in the treatment of symptomatic knee osteoarthritis (OA). Methods. The study was a randomized, double-blind, placebo-controlled, proof-of-concept trial. Intraarticular...... in joint space width (JSW) seen on radiographs, and pain scores on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Results. One hundred ninety-two patients were randomized and evaluated for safety, 180 completed the trial, and 168 were evaluated for the primary efficacy end...

  10. Two insulin-like growth factor I messenger RNAs are expressed in human liver.

    OpenAIRE

    Rotwein, P

    1986-01-01

    Through use of a synthetic oligonucleotide probe, human insulin-like growth factor I (IGF-I) cDNA clones were isolated from a liver library. Two types of cDNAs were defined by restriction enzyme analysis and DNA sequencing. Both encode IGF-I precursors of either 195 or 153 amino acids. The two predicted protein precursors are identical from their amino terminus to a lysine residue 16 codons beyond the IGF-I sequence, and then they diverge. Both cDNAs predict additional unique carboxyl-termina...

  11. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

    OpenAIRE

    1984-01-01

    The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to th...

  12. Effects of recombinant human insulin-like growth factor I on glomerular dynamics in the rat.

    OpenAIRE

    Hirschberg, R; Kopple, J D; Blantz, R C; Tucker, B J

    1991-01-01

    This study was undertaken to investigate the mechanisms by which an infusion of recombinant human insulin-like growth factor I (rhIGF-I) increases GFR and renal plasma flow (RPF) in rats. Glomerular micropuncture studies were carried out in 14 nonstarved Munich Wistar rats and in 12 rats deprived of food for 60-72 h. Animals were given an intravenous injection and infusion of either rhIGF-I or vehicle. In both nonstarved and starved animals, the IGF-I injection and infusion increased the seru...

  13. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

    Directory of Open Access Journals (Sweden)

    J.C. Zhang

    2014-10-01

    Full Text Available Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs expressing human basic fibroblast growth factor (hbFGF. After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC, MSCs expressing hbFGF (hbFGF-MSC, MSC controls, and phosphate-buffered saline (PBS controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001; however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008 and microvessel density (P<0.001. Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

  14. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, J.C. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Zheng, G.F. [Department of Vascular Surgery, The People' s Hospital of Ganzhou, Ganzhou (China); Wu, L.; Ou Yang, L.Y.; Li, W.X. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-08-08

    Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs) expressing human basic fibroblast growth factor (hbFGF). After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC), MSCs expressing hbFGF (hbFGF-MSC), MSC controls, and phosphate-buffered saline (PBS) controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF) expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001); however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008) and microvessel density (P<0.001). Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

  15. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani;

    2015-01-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...... such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure...

  16. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.

    Science.gov (United States)

    Rauchenberger, Robert; Borges, Eric; Thomassen-Wolf, Elisabeth; Rom, Eran; Adar, Rivka; Yaniv, Yael; Malka, Michael; Chumakov, Irina; Kotzer, Sarit; Resnitzky, Dalia; Knappik, Achim; Reiffert, Silke; Prassler, Josef; Jury, Karin; Waldherr, Dirk; Bauer, Susanne; Kretzschmar, Titus; Yayon, Avner; Rothe, Christine

    2003-10-03

    The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

  17. Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

    Science.gov (United States)

    Chua, K H; Aminuddin, B S; Fuzina, N H; Ruszymah, B H I

    2004-05-01

    We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

  18. Apoptosis of Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-p2 in vitro

    Institute of Scientific and Technical Information of China (English)

    CAO Yang(曹 阳); WEI Houren(魏厚仁); Pfaffl Michael; DA Banghong(笪邦红); LI Zhongyu(李忠玉)

    2004-01-01

    Summary: Whether transforming growth factor-β2 (TGF-β2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-β2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy.DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43±1.17) % and (9. 60±2.05) % respectively with different concentrations [1 ng/ml (P<0. 05), 3.2 ng/ml (P<0.01)] of TGF-β2 with the difference being significant between experimental group and control group[(1. 41±0.34) %]. It was concluded that TGF-β2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.

  19. 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...... as in a few Paneth cells. Glandular structures, such as the glands of the cardia and the pyloric part of the stomach, Brunner's glands of the duodenum, the pancreas, and the submucous glands of the trachea, showed positive EGF immunoreactivity later (week 17). Thus, apart from the kidney, staining...

  20. Purification and characterization of an insulin-like growth factor II variant from human plasma.

    Science.gov (United States)

    Hampton, B; Burgess, W H; Marshak, D R; Cullen, K J; Perdue, J F

    1989-11-15

    An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.

  1. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

    Directory of Open Access Journals (Sweden)

    Fatemeh Kazemi-Lomedasht

    2014-12-01

    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  2. Safety of recombinant human platelet-derived growth factor-BB in Augment® Bone Graft

    Directory of Open Access Journals (Sweden)

    Luis A Solchaga

    2012-12-01

    Full Text Available This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment® Bone Graft (Augment. Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

  3. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  4. Parotid gland is the main source of human salivary epidermal growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Thesleff, I.; Viinikka, L.; Saxen, L.; Lehtonen, E.; Perheentupa, J.

    1988-01-01

    To clarify the production of human epidermal growth factor (EGF) by different salivary glands, the authors measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, they studied the presence of EGF in PG and SMG by immunohistochemistry. The mean concentrations of EDG in PG saliva was higher than in whole saliva, which in turn was higher than in mixed SMG + SLG saliva. No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, buy only in PG there were prominent EDG deposits in luminal spaces. Their data suggest that EDG is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EDG is mainly from SMG.

  5. [Association of the insulin-like growth factor II (IGF2) gene with human cognitive functions].

    Science.gov (United States)

    Alfimova, M V; Lezheĭko, T V; Gritsenko, I K; Golimbet, V E

    2012-08-01

    Active search for candidate genes whose polymorphisms are associated with human cognitive functions has been in progress in the past years. The study focused on the role that the insulin-like growth factor II (IGF2) gene may play in the variation of cognitive processes related to executive functions. The ApaI polymorphism of the IGF2 gene was tested for association with selective attention during visual search, working memory/mental control, and semantic verbal fluency in a group of 182 healthy individuals. The ApaI polymorphism was associated with the general cognitive index and selective attention measure. Carriers of genotype AA displayed higher values of the two parameters than carriers of genotype GG. It was assumed that the ApaI polymorphism of the IGF2 gene influences the human cognitive functions, acting possibly via modulation of the IGF-II level in the central nervous system.

  6. Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat

    Institute of Scientific and Technical Information of China (English)

    RONG Shu-Ling; LU Yong-Xin; LIAO Yu-Hua; WANG Xiao-Lin; GUO He-Ping; CHANG Chao; GAO Yan-Zhang; MI Shao-Hua; Wan Jian-Ping

    2006-01-01

    Background This study transferred a recombinant gene encoding human insulin like growth factor-1 (hIGF-1)into modified primary skeletal myoblasts with a retroviral vector (pLgXSN) and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hIGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochemistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghIGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth.Results Recombinant retroviral plasmid vector pLghIGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening. hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast (P< 0.05). The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast (P< 0.05). Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myoblast group (P< 0.05).Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.

  7. Radioimmunoassay of a human serum growth factor for Balb/c-3T3 cells: derivation from platelets.

    Science.gov (United States)

    Antoniades, H N; Scher, C D

    1977-05-01

    A radioimmunoassay has been developed for the detection and quantification of a human serum polypeptide that has growth-promoting activity for confluent Balb/c-3T3 cells. Antiserum to this growth factor does not recognize antigens in mouse, guinea pig, or bovine serum but does detect some crossreacting antigen in the serum of the New World monkey Cebus albifrons and more in the serum of the Old World rhesus monkeys Macaca mulatta and M. fascicularis, demonstrating that the antigenic determinants of the growth factor have a degree of species specificity. Serum derived from whole human blood contains approximately 770 pg of the growth factor per mg of protein; serum derived from platelet-poor blood contains about 112 pg of the growth factor per mg of protein. As much as 1 microng of the growth factor per mg of protein has been recovered from human platelets by heating them at 100 degrees for 2 min. Approximately 1-2 ng of the growth factor, in either whole serum or platelets, stimulates 5 to 10 X 10(3) confluent Balb/c-3T3 cells to replicate. The heat treatment of platelets allows the purification and quantitative recovery of the growth factor from blood.

  8. Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

    Institute of Scientific and Technical Information of China (English)

    龚振宇; 周树夏; 顾晓明; 李涤尘; 孙明林

    2003-01-01

    Objective: To investigate the effect of recombinant human basic fibroblast growth factor (rhbFGF) on angiogenesis during mandible fracture healing in rabbit. Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor VIII related antigen (F8-RA) in callus was examined with immunohistochemical staining. Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P<0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.

  9. Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations%Therapeutic targeting of epidermal growth factor receptor in humancancer: successes and limitations

    Institute of Scientific and Technical Information of China (English)

    Jill Wykosky; Tim Fenton; Frank Furnari; Webster K. Cavenee

    2011-01-01

    Epidermal growth factor receptor (EGFR) is one of the most commonly altered genes in human cancer by way of over-expression, amplification, and mutation. Targeted inhibition of EGFR activity suppresses signal transduction pathways which control tumor cell growth, proliferation, and resistance to apoptosis. Small molecule tyrosine kinase inhibitors and monoclonal antibodies are among the most common EGFR-targeting agents and have been used clinically for treating various malignancies. This review discusses the successes and challenges of targeting EGFR in human cancer. The genetic alterations of EGFR tend to occur more often in some solid tumors than others, as do the mechanisms of resistance to targeted inhibition. The clinical and basic science experiences with these agents thus far have important implications for the future of therapeutic targeting of EGFR.

  10. Brain-derived neurotrophic factor increases vascular endothelial growth factor expression and enhances angiogenesis in human chondrosarcoma cells.

    Science.gov (United States)

    Lin, Chih-Yang; Hung, Shih-Ya; Chen, Hsien-Te; Tsou, Hsi-Kai; Fong, Yi-Chin; Wang, Shih-Wei; Tang, Chih-Hsin

    2014-10-15

    Chondrosarcomas are a type of primary malignant bone cancer, with a potent capacity for local invasion and distant metastasis. Brain-derived neurotrophic factor (BDNF) is commonly upregulated during neurogenesis. The aim of the present study was to examine the mechanism involved in BDNF-mediated vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells. Here, we knocked down BDNF expression in chondrosarcoma cells and assessed their capacity to control VEGF expression and angiogenesis in vitro and in vivo. We found knockdown of BDNF decreased VEGF expression and abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in vivo in the chick chorioallantoic membrane and Matrigel plug nude mouse models. In addition, in the xenograft tumor angiogenesis model, the knockdown of BDNF significantly reduced tumor growth and tumor-associated angiogenesis. BDNF increased VEGF expression and angiogenesis through the TrkB receptor, PLCγ, PKCα, and the HIF-1α signaling pathway. Finally, we analyzed samples from chondrosarcoma patients by immunohistochemical staining. The expression of BDNF and VEGF protein in 56 chondrosarcoma patients was significantly higher than in normal cartilage. In addition, the high level of BDNF expression correlated strongly with VEGF expression and tumor stage. Taken together, our results indicate that BDNF increases VEGF expression and enhances angiogenesis through a signal transduction pathway that involves the TrkB receptor, PLCγ, PKCα, and the HIF-1α. Therefore, BDNF may represent a novel target for anti-angiogenic therapy for human chondrosarcoma.

  11. Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression.

    Science.gov (United States)

    Hojo, Y; Ikeda, U; Maeda, Y; Takahashi, M; Takizawa, T; Okada, M; Funayama, H; Shimada, K

    2000-05-01

    The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.

  12. Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Kazuya Kitamura

    Full Text Available Many therapeutic interventions for spinal cord injury (SCI using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF, which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.

  13. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    Institute of Scientific and Technical Information of China (English)

    Run-Ping Gao; David R Brigstock

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-b1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen Ⅰ, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen Ⅰ, and an increase in produced and secreted CCN2 or extracellular collagen Ⅰ protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen Ⅰ protein. Furthermore, the TGF-b1-induced increase in mRNA or protein for CCN2 or collagen Ⅰ was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-b1-induced collagen Ⅰ production in human HSCs and regulates entry of the cells into Sphase.

  14. Placental growth factor is a potent vasodilator of rat and human resistance arteries.

    Science.gov (United States)

    Osol, George; Celia, Gerard; Gokina, Natalia; Barron, Carolyn; Chien, Edward; Mandala, Maurizio; Luksha, Leonid; Kublickiene, Karolina

    2008-03-01

    The objectives of this study were to determine whether placental growth factor (PlGF) exerts a vasodilatory effect on rat uterine vessels (arcuate arteries and veins) and to examine regional differences in reactivity by comparing these responses to those of comparably sized mesenteric vessels. We also sought to examine and compare its effects on human uterine and subcutaneous vessels. All vessels were studied in vitro, under pressurized (rat) or isometric wire-mounted (human) conditions, and exposed to a range of PlGF concentrations. Inhibitors of nitric oxide and prostaglandin synthesis were included in an effort to understand the causal mechanism(s). In rat uterine arteries, the effects of receptor inhibition and activation using selective ligands for VEGFR-1 (PlGF) vs. VEGFR-2 (VEGF-E) were determined, and real-time RT-PCR was performed to evaluate the effect of pregnancy on relative abundance of VEGFR-1 and VEGFR-2 message in the vascular wall. PlGF was a potent vasodilator of all vessels studied, with greatest sensitivity observed in rat uterine arteries. Pregnancy significantly augmented dilator sensitivity to PlGF, and this effect was associated with selective upregulation of VEGFR-1 message in the pregnant state. The contribution of nitric oxide was appreciable in rat and human uterine arteries, with lesser effects in rat uterine veins and mesenteric arteries, and with no observable effect in human subcutaneous vessels. Based on these results, we conclude that PlGF is a potent vasodilator of several vessel types in both humans and rats. Its potency and mechanism vary with physiological state and vessel location and are mediated solely by the VEGFR-1 receptor subtype. Gestational changes in the uterine circulation suggest that this factor may play a role in modulating uterine vascular remodeling and blood flow during the pregnant state.

  15. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    Science.gov (United States)

    Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

    2012-01-01

    Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility

  16. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Kawamura

    Full Text Available Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for

  17. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle.

    Science.gov (United States)

    Hoier, Birgitte; Prats, Clara; Qvortrup, Klaus; Pilegaard, Henriette; Bangsbo, Jens; Hellsten, Ylva

    2013-09-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P<0.05) after exercise. The observation of more VEGF vesicles close to sarcolemma after exercise, combined with a 5-fold increase (P<0.05) in VEGF in the interstitial fluid, suggest that VEGF-containing vesicles redistribute to sarcolemma and that VEGF is secreted to the extracellular fluid. This study provides the first evidence in humans for a mechanism by which skeletal muscle fibers can control capillary growth by releasing VEGF from intracellular vesicles during contraction.

  18. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    Science.gov (United States)

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  19. Human insulin-like growth factor II leader 2 mediates internal initiation of translation

    DEFF Research Database (Denmark)

    Pedersen, Susanne K; Christiansen, Jan; Hansen, Thomas v O

    2002-01-01

    Insulin-like growth factor II (IGF-II) is a fetal growth factor, which belongs to the family of insulin-like peptides. During fetal life, the IGF-II gene generates three mRNAs with different 5' untranslated regions (UTRs), but identical coding regions and 3' UTRs. We have shown previously that IG...

  20. Basic fibroblast growth factor and its receptors in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Ales Hampl

    2005-12-01

    Full Text Available Human embryonic stem cells (hESCs are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs, which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.

  1. Recombinant expression of human nerve growth factor beta in rabbit bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Fan, Bo-Sheng; Lou, Ji-Yu

    2010-12-01

    Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In the present study, the recombinant expression of human nerve growth factor beta (hNGF-β) gene in rabbit bone marrow mesenchymal stem cells (rMSCs) was undertaken. Recombinant vector containing hNGF-β was constructed and transferred into rMSCs, the expressions of the exogenous in rMSCs were determined by reverse transcriptase PCR (RT-PCR), ELISA and Western blot, whereas the biological activity of recombinant hNGF-β was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. The results showed that the hNGF-β gene expressed successfully in the rMSCs, a polypeptide with a molecular weight of 13.2 kDa was detected. The maximal expression level of recombinant hNGF-β in rMSCs reached 126.8012 pg/10(6) cells, the mean concentration was 96.4473 pg/10(6) cells. The recombinant hNGF-β in the rMSCs showed full biological activity when compared to commercial recombinant hNGF-β.

  2. Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy.

    Science.gov (United States)

    Sun, Guilian; Haginoya, Kazuhiro; Wu, Yanling; Chiba, Yoko; Nakanishi, Tohru; Onuma, Akira; Sato, Yuko; Takigawa, Masaharu; Iinuma, Kazuie; Tsuchiya, Shigeru

    2008-04-15

    The detailed process of how dystrophic muscles are replaced by fibrotic tissues is unknown. In the present study, the immunolocalization and mRNA expression of connective tissue growth factor (CTGF) in muscles from normal and dystrophic human muscles were examined with the goal of elucidating the pathophysiological function of CTGF in muscular dystrophy. Biopsies of frozen muscle from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, spinal muscular atrophy, congenital myopathy were analyzed using anti-CTGF polyclonal antibody. Reverse transcription-polymerase chain reaction (RT-PCR) was also performed to evaluate the expression of CTGF mRNA in dystrophic muscles. In normal muscle, neuromuscular junctions and vessels were CTGF-immunopositive, which suggests a physiological role for CTGF in these sites. In dystrophic muscle, CTGF immunoreactivity was localized to muscle fiber basal lamina, regenerating fibers, and the interstitium. Triple immunolabeling revealed that activated fibroblasts were immunopositive for CTGF and transforming growth factor-beta1 (TGF-beta1). RT-PCR analysis revealed increased levels of CTGF mRNA in the muscles of DMD patients. Co-localization of TGF-beta1 and CTGF in activated fibroblasts suggests that CTGF expression is regulated by TGF-beta1 through a paracrine/autocrine mechanism. In conclusion, TGF-beta1-CTGF pathway may play a role in the fibrosis that is commonly observed in muscular dystrophy.

  3. Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

    Directory of Open Access Journals (Sweden)

    Sonia Covaceuszach

    Full Text Available Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab. The humanized antibody (hum-αD11 was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

  4. Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

    Science.gov (United States)

    Covaceuszach, Sonia; Marinelli, Sara; Krastanova, Ivet; Ugolini, Gabriele; Pavone, Flaminia; Lamba, Doriano; Cattaneo, Antonino

    2012-01-01

    Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates. In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles. PMID:22403636

  5. Mitogenic properties of insulin-like growth factors I and II, insulin-like growth factor binding protein-3 and epidermal growth factor on human breast epithelial cells in primary culture.

    Science.gov (United States)

    Strange, Karen S; Wilkinson, Darcy; Emerman, Joanne T

    2002-10-01

    Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) are growth factors implicated in mammary gland development and are believed to be involved in breast cancer. However, the interactions between components of the IGF system and breast epithelial cells, which give rise to breast cancer, are not well understood. We have investigated the mitogenic properties of IGF-I, IGF-II, IGF binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast epithelial cells (HBEC) in primary culture. We show that, under serum-free conditions, HBEC are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of HBEC growth in primary culture and also associated with breast cancer, appear to stimulate HBEC in a synergistic manner. IGFBP-3 inhibits the stimulation by IGF-I, IGF-II and IGF-I plus EGE In addition, it appears that IGFBP-3 has an inhibitory effect on HBEC growth that is IGF-independent. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on HBEC growth in primary culture. Characterizing the role of the IGF system in normal breast biology is significant because the system has been implicated in breast cancer and a number of the anti-estrogens used in treatment are believed to function through the IGF system.

  6. Comparative study of the effects of recombinant human epidermal growth factor and basic fibroblast growth factor on corneal epithelial wound healing and neovascularization in vivo and in vitro.

    Science.gov (United States)

    Yan, Limeng; Wu, Wei; Wang, Zhichong; Li, Chaoyang; Lu, Xiaohe; Duan, Hucheng; Zhou, Jin; Wang, Xiaoran; Wan, Pengxia; Song, Yiyue; Tang, Jing; Han, Yu

    2013-01-01

    This study was undertaken to investigate the effects of recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) on corneal wound healing and neovascularization (CNV). The positive effects of 10 ng/ml rhEGF and bFGF on the proliferation of corneal epithelial cells (SD-HCEC1s), rabbit keratocyte cells (RKCs) and human umbilical vein endothelial cells (HUVECs) as well as the effects on the migration capacity on HUVECs were observed. An animal central corneal wound and CNV model was established in rabbits. One eye of each group was chosen randomly for topical administration of rhEGF, bFGF or normal saline, and variability in the area of corneal epithelial wound healing and CNV was observed. The optimal concentration of rhEGF and bFGF for the proliferation of corneal epithelial cells was 10 ng/ml. The promotive effect of 10 ng/ml rhEGF on the proliferation of RKCs and HUVECs was less than that of 10 ng/ml bFGF. In the animal experiment, the healing rate of the corneal epithelium in the rhEGF group was better than in the other groups on day 1. On day 3, the healing rates of the 3 groups were nearly equal. The CNV area in the rhEGF group was less than that of the bFGF group. rhEGF and bFGF both had promotive effects on corneal epithelial wound healing, but rhEGF had a weaker promotive effect on CNV than bFGF. With long-term application of growth factor drugs, rhEGF is suggested for lessening the growth of CNV. Copyright © 2012 S. Karger AG, Basel.

  7. Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

    Science.gov (United States)

    Kagel, J R; Rossi, D T; Nordblom, G D; Dudeck, R C; Barksdale, C M; Kuo, B S; Wright, D S

    1995-09-01

    A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

  8. Differential regulation of type I interferon and epidermal growth factor pathways by a human Respirovirus virulence factor.

    Directory of Open Access Journals (Sweden)

    Grégory Caignard

    2009-09-01

    Full Text Available A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C. We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E. Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.

  9. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Wang Jianing

    2011-05-01

    Full Text Available Abstract Background The human coagulation trigger tissue factor (TF is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. Methods The specific small interfering RNA (siRNA designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. Results TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. Conclusions Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

  10. Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

    OpenAIRE

    Bayne, M L; Cascieri, M A; Kelder, B; Applebaum, J; Chicchi, G; Shapiro, J A; Pasleau, F.; Kopchick, J. J.

    1987-01-01

    A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fi...

  11. Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia

    DEFF Research Database (Denmark)

    Hojman, Pernille; Pedersen, Maria; Nielsen, Anders Rinnov

    2009-01-01

    OBJECTIVE: Fibroblast growth factor-21 (FGF-21) is a potent metabolic regulator, which in animal models has been shown to improve glucose metabolism and insulin sensitivity. Recently, FGF-21 was shown to be expressed and secreted from murine muscle cells in response to insulin stimulation. RESEARCH...... DESIGN AND METHODS: We studied muscular FGF-21 expression and plasma FGF-21 after acute insulin stimulation in young healthy men during a hyperinsulinemic-euglycemic clamp. Furthermore, we investigated systemic levels and muscle FGF-21 expression in humans with or without insulin resistance and chronic...... elevated insulin. RESULTS: FGF-21 was barely detectable in young healthy men before insulin infusion. After 3 or 4 h of insulin infusion during a hyperinsulinemic-euglycemic clamp, muscular FGF-21 expression increased significantly. Plasma FGF-21 followed the same pattern. In individuals with chronic...

  12. An ultrasensitive time-resolved immunofluorometric assay of human epidermal growth factor.

    Science.gov (United States)

    Pesonen, K; Alfthan, H; Stenman, U H; Viinikka, L; Perheentupa, J

    1986-09-01

    We have developed a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for human epidermal growth factor (hEGF) in body fluids. A two-step solid-phase technique was used. The assay utilizes a polyclonal anti-hEGF attached to the solid phase, and a monoclonal anti-hEGF labeled with Europium (III) as a tracer. The sensitivity of the assay (2.5 pg/ml) is at least 20 times better than what has been achieved by radioimmunoassay (RIA), and the measuring range is much wider: 2.5-5000 pg/ml. The feasibility of TR-IFMA was tested by assaying urine containing large amounts and amniotic fluid containing small amounts (mostly undetectable by RIA) of immunoreactive hEGF. The correlation between urine hEGF concentrations (1-100 ng/ml) measured by RIA and TR-IFMA was good: r = 0.96.

  13. Analysis of human transforming growth factor β-induced gene mutation in corneal dystrophy

    Institute of Scientific and Technical Information of China (English)

    李杨; 孙旭光; 任慧媛; 董冰; 王智群; 孙秀英

    2004-01-01

    Background Corneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy. Methods Three families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor β-induced gene (BIGH3 gene) was performed. Results Five individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H. Conclusion Molecular genetic analysis can improve accurate diagnosis of corneal dystrophy.

  14. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  15. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...... of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal...... regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P...

  16. Differential Regulation of Human Thymosin Beta 15 Isoforms by Transforming Growth Factor Beta 1

    Science.gov (United States)

    Banyard, Jacqueline; Barrows, Courtney; Zetter, Bruce R.

    2009-01-01

    We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080 and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322 and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility. PMID:19296525

  17. Epidermal growth factor suppresses insulin-like growth factor binding protein 3 levels in human papillomavirus type 16-immortalized cervical epithelial cells and thereby potentiates the effects of insulin-like growth factor 1.

    Science.gov (United States)

    Hembree, J R; Agarwal, C; Eckert, R L

    1994-06-15

    Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b

  18. Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

    Directory of Open Access Journals (Sweden)

    Corleta H.

    2000-01-01

    Full Text Available Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1 interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5% followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

  19. A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone).

    Science.gov (United States)

    Hayashi, K; Nomoto, H; Kurobe, M; Nishimuro, S; Hiratani, H; Furukawa, S

    1985-06-01

    A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.

  20. Regulation Effect of Vascular Endothelial Growth Factor on Human Fetal Choroid Vascularization

    Institute of Scientific and Technical Information of China (English)

    JinsongZhao; YiWang; 等

    2002-01-01

    Purpose:To investigate the spatial and temporal regulation effect of vascular endothelial growth factor(VEGF) on human fetal choroids vascularization.Methods:The eyeballs of 54 human fetuses from the 9th week to the 40th week due to accidental abortion were studied by immunohistochemically stainin for the expression of VEGF and proliferation cell nuclear antigen (PCNA).Results: (1)The distribution of VEGF expression in the retinal pigment epithelium (RPE) decreased with the incrase of age,the peak of which was between the 9th and 14th week.(2)PCNA immunoreactivity was localized within choriocapillaris endothelium .The expression level decreased alone with fetus age.In this period the choriocapillaris endothelium kept proliferation,differentiation,canalization and remodeled to form the choroids vessels(3)Statistically significant correlations were shown between the expression of VEGF in the PRE and that of PCNA in choriocapillaris endothelium(r=0.933,P<0.01).Couclusin:VEGF expression in PRE was positively involved in modulating human fetal choroids vascularization .Eye Science 2000;16:11-14.

  1. Regulation Effect of Vascular Endothelial Growth Factor on Human Fetal Choroid Vascularization

    Institute of Scientific and Technical Information of China (English)

    Jinsong Zhao; Yue Song; Yi Wang; Xiaoguang Zhang

    2000-01-01

    Purpose: To investigate the spatial and temporal regulation effect of vascular endothelial growth factor(VEGF) on human fetal choroid vascularization. Methods: The eyeballs of 54 human fetuses from the 9th week to the 40th week due to accidental abortion were studied by immunohistochemically staining for the expression of VEGF and proliferation cell nuclear antigen (PCNA). Results: (1) The distribution of VEGF expression in the retinal pigment epithelium (RPE) decreased with the increase of age, the peak of which was between the 9th and 14th week. (2) PCNA immunoreactivity was localized within choriocapillaris endothelium. The expression level decreased alone with fetus age. In this period the choriocapillaris endothelium kept proliferation, differentiation, canalization and remodelled to form the choroid vessels. (3)Statistically significant correlations were shown between the expression of VEGF in the PRE and that of PCNA in choriocapillaris endothelium(r =0. 933, P < 0. 01). Conclusion: VEGF expression in RPE was positively involved in modulating human fetal choroid vascularization. Eye Science 2000; 16:11 ~ 14.

  2. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    Science.gov (United States)

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A

    1995-01-01

    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  3. Learning and memory changes in rats following exogenous human hepatocyte growth factor gene injection into cerebral ischemic penumbra

    Institute of Scientific and Technical Information of China (English)

    Zhijun You; Yong Liu; Jianye Yang; Qingping Jiang

    2011-01-01

    Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene expression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.

  4. Expression of transforming growth factor-β2in vitreous body and adjacent tissues during prenatal development of human eye.

    Science.gov (United States)

    Sukhikh, G T; Panova, I G; Smirnova, Yu A; Milyushina, L A; Firsova, N V; Markitantova, Yu V; Poltavtseva, R A; Zinov'eva, R D

    2010-12-01

    Expression of transforming growth factor-β2 was detected by PCR in the vitreous body, lens, retina, and ciliary-iris complex of human eye at early stages of fetal development. Immunochemical assay of the corresponding protein in eye tissues revealed a correlation between the localization of transforming growth factor-β2 and the development of intraocular hyaloid vascular network, its regression, formation of the vitreous body, and development of definite retinal vessels.

  5. Upregulation of vascular endothelial growth factor by hydrogen peroxide in human colon cancer

    Institute of Scientific and Technical Information of China (English)

    Jian-Wei Zhu; Bao-Ming Yu; Yu-Bao Ji; Ming-Hua Zheng; Dong-Hua Li

    2002-01-01

    AIM: To evaluate the effect of reactive oxygen species suchas hydrogen peroxide on the progression of human coloncancer.METHODS: Human colon carcinoma cell lines, LS174T andHCT8, were treated respectively with 10- 5,10- 7 or 10- 9 mol@L- 1 hydrogen peroxide for 24h, and co-cultured with humanendothelial cell line ECV-304. The migration of ECV-304induced by cancer cells was calculated and the expressionlevel of vascular endothelial growth factor in cancer cellswas determined by RT-PCR analysis and ELISA.Dactinomycin of 1.5mg@ L-1 which could block transcriptionof cancer cells was applied to observe the effects of H2O2 ontranscriptional activity and the relative half-life of VEGFmRNA. Finally, to evaluate the effect H2O2 on NF-κB activityin colon cancer cells, NF-κB in cytoplasm and nucleus of thecells were detected with FITC-tagged antibody and itspresence in the nucleus (Fn ) Vs cytoplasm ( Fc ) wasmonitored by measuring the green fluorescence integratedover the nucleus by laser scanning cytometry(LSC).RESULTS: Exogenouse hydrogen peroxide of lowconcentration increased the migration of endothelial cellsinduced by colon cancer cells. When cancer cells weretreated with 10-5 mol@ L-1 H2O2, the migration number ofendothelial cells induced by LS174T cells was 203 ± 70, andthe number induced by HCT8 cells was 145 ± 65. The twovalues were significantly higher than those treated with otherconcentrations of h2O2 ( P < 0.01 ). The expression ofvascular endothelial growth factor in cancer cells, whichcould be blocked by dactinomycin, were increased to acertain degree, while the relative half-life of VEGF mRNAwas not prolonged after treatment with hydrogen peroxide.The activity of NF-κB in colon cells rose after the cells wereexposed to hydrogen peroxide for 24h. The Fn values inHCT8 cells were 91 ± 13 (0 mol@ L- 1 h2O2) and 149 ± 40( 10-5mol@L-1 h2O2) ( P< 0.05), in LS174T cells were 127 ± 35(0mol@L-1 H2O2) and 192± 11(10-5 mol@L-1 h2O2) (P< 0.05).lt is similar

  6. Hypertrophy of neurons within cardiac ganglia in human, canine, and rat heart failure: the potential role of nerve growth factor.

    Science.gov (United States)

    Singh, Sanjay; Sayers, Scott; Walter, James S; Thomas, Donald; Dieter, Robert S; Nee, Lisa M; Wurster, Robert D

    2013-08-19

    Autonomic imbalances including parasympathetic withdrawal and sympathetic overactivity are cardinal features of heart failure regardless of etiology; however, mechanisms underlying these imbalances remain unknown. Animal model studies of heart and visceral organ hypertrophy predict that nerve growth factor levels should be elevated in heart failure; whether this is so in human heart failure, though, remains unclear. We tested the hypotheses that neurons in cardiac ganglia are hypertrophied in human, canine, and rat heart failure and that nerve growth factor, which we hypothesize is elevated in the failing heart, contributes to this neuronal hypertrophy. Somal morphology of neurons from human (579.54±14.34 versus 327.45±9.17 μm(2); Phypertrophy of neurons in cardiac ganglia compared with controls. Western blot analysis shows that nerve growth factor levels in the explanted, failing human heart are 250% greater than levels in healthy donor hearts. Neurons from cardiac ganglia cultured with nerve growth factor are significantly larger and have greater dendritic arborization than neurons in control cultures. Hypertrophied neurons are significantly less excitable than smaller ones; thus, hypertrophy of vagal postganglionic neurons in cardiac ganglia would help to explain the parasympathetic withdrawal that accompanies heart failure. Furthermore, our observations suggest that nerve growth factor, which is elevated in the failing human heart, causes hypertrophy of neurons in cardiac ganglia.

  7. The relationship between human resource development factors, career growth and turnover intention: The mediating role of organizational commitment

    Directory of Open Access Journals (Sweden)

    Muhammad Shahid Nawaz

    2016-02-01

    Full Text Available Retaining the best employees is of high concern for most organizations and this issue has become a significant focus of attention for many researchers. For this reason, this paper discusses different factors which influence the employee turnover intention-behavior in the organization, specifically to examine the effect of salary, performance appraisal, training & development and career growth on turnover intention. In addition, based on the social exchange theory this paper explains the mediating role of organizational commitment in the relationship between human resource development factors, career growth and turnover intention. A cross sectional, survey data study is undertaken to investigate the relationships in a sample of 270 full time faculty members employed in different private universities of Pakistan. Partial Least Square two step path modeling is used to test the direct and the indirect hypothesis of the study. The results of PLS (SEM path modeling reveal that human resource development factors specially salary and performance appraisal were negatively associated with turnover intention. In addition, the results also indicate that career growth had significant relationships with turnover intention. Moreover, out of four dimensions of career growth, only two dimensions, namely promotion speed and remuneration growth, have strong influence on turnover intention. Finally, in terms of organizational commitment as mediating variable between the relationships of salary, performance appraisal, career growth and turnover intention, four out of six variables indicate partial mediation including career growth (career goal progress, career growth (promotion speed, career growth (remuneration growth and performance appraisal.

  8. The portal-drained viscera release fibroblast growth factor 19 in humans.

    Science.gov (United States)

    Koelfat, Kiran V K; Bloemen, Johanne G; Jansen, Peter L M; Dejong, Cornelis H C; Schaap, Frank G; Olde Damink, Steven W M

    2016-12-01

    Fibroblast growth factor 19 (FGF19) is an ileum-derived endrocrine factor that is produced in response to transepithelial bile salt flux. FGF19 represses bile salt synthesis in the liver. Despite the general assumption that FGF19 signals to the liver via portal blood, no human data are available to support this notion. The aim was to study portal FGF19 levels, and determined bile salt and FGF19 fluxes across visceral organs in humans. Bile salt and FGF19 levels were assessed in arterial, portal, and hepatic venous blood collected from fasted patients who underwent partial liver resection for colorectal liver metastases (n = 30). Fluxes across the portal-drained viscera (PDV), liver, and splanchnic area were calculated. Portal bile salt levels (7.8 [5.0-12.4] μmol/L) were higher than levels in arterial (2.7 [1.7-5.5] μmol/L, P FGF19 (161 ± 78 pg/mL) were higher than arterial levels (135 ± 65 pg/mL, P = 0.046). A net release of FGF19 by the PDV (+4.0 [+2.1 to +9.9] ng kg(-1) h(-1), P FGF19 across the liver (-0.2 [-3.7 to +7.4] ng kg(-1) h(-1), P = 0.93). In conclusion, FGF19 levels in human portal blood are higher than in arterial blood. FGF19 is released by the portal-drained viscera under fasted steady state conditions.

  9. Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

    Science.gov (United States)

    Kono, Kiyomi; Maeda, Hidefumi; Fujii, Shinsuke; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Monnouchi, Satoshi; Teramatsu, Yoko; Hamano, Sayuri; Koori, Katsuaki; Akamine, Akifumi

    2013-05-01

    Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

  10. 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-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.

  11. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

    Science.gov (United States)

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-11-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

  12. Differential gene expression profiling of human epidermal growth factor receptor 2-overexpressing mammary tumor

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Haining Peng; Yingli Zhong; Daiqiang Li; Mi Tang; Xiaofeng Ding; Jian Zhang

    2008-01-01

    Human epidermal growth factor receptor 2 (HER2) is highly expressed in approximately 30% of breast cancer patients,and substantial evidence supports the relationship between HER2 overexpression and poor overall survival. However,the biological function of HER2 signaltransduction pathways is not entirely clear. To investigate gene activation within the pathways, we screened differentially expressed genes in HER2-positive mouse mammary tumor using two-directional suppression subtractive hybridization combined with reverse dot-blotting analysis. Forty genes and expressed sequence tags related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins were differentially expressed in HER2-positive mammary tumor tissue. Among these, 19 were already reported to be differentially expressed in mammary tumor, 11 were first identified to be differentially expressed in mammary tumor in this study but were already reported in other tumors, and 10 correlated with other cancers. These genes can facilitate the understanding of the role of HER2 signaling in breast cancer.

  13. 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.

  14. Expression of bioactive recombinant human fibroblast growth factor 10 in Carthamus tinctorius L. seeds.

    Science.gov (United States)

    Huang, Jian; Yang, Jing; Guan, Lili; Yi, Shanyong; Du, Linna; Tian, Haishan; Guo, Yongxin; Zhai, Feng; Lu, Zhen; Li, Haiyan; Li, Xiaokun; Jiang, Chao

    2017-10-01

    Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein. Copyright © 2016. Published by Elsevier Inc.

  15. The growth inhibition of human breast cancer cells by a novel synthetic progestin involves the induction of transforming growth factor beta.

    OpenAIRE

    Colletta, A. A.; Wakefield, L M; Howell, F. V.; Danielpour, D; Baum, M.; Sporn, M B

    1991-01-01

    Recent experimental work has identified a novel intracellular binding site for the synthetic progestin, Gestodene, that appears to be uniquely expressed in human breast cancer cells. Gestodene is shown here to inhibit the growth of human breast cancer cells in a dose-dependent fashion, but has no effect on endocrine-responsive human endometrial cancer cells. Gestodene induced a 90-fold increase in the secretion of transforming growth factor-beta (TGF-beta) by T47D human breast cancer cells. O...

  16. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

    Science.gov (United States)

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

  17. Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors.

    Science.gov (United States)

    Valve, E; Martikainen, P; Seppänen, J; Oksjoki, S; Hinkka, S; Anttila, L; Grenman, S; Klemi, P; Härkönen, P

    2000-12-01

    FGF-8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF-8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF-8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF-8 is over-expressed in human breast and prostate cancers. Expression has also been found in RT-PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF-8 isoforms are expressed in ovarian cancer and whether FGF-8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT-PCR and immunohistochemistry. FGF-8 isoform b was expressed in all ovarian tumors and in all 7 ovarian-cancer cell lines studied. Isoform a was co-expressed in 9 malignant ovarian tumors. FGF-8 mRNA was not detected by RT-PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF-8 protein to cancer cells. In general, the increased intensity of FGF-8 staining was associated with loss of differentiation within the tumors (Bowker's test, p = 0.37). FGF-8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT-PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF-8 receptors expressed in normal ovaries and in ovarian tumors. FGF-8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF-8 have acquired the capacity of expressing FGF-8. This suggests that FGF-8 has an important role in ovarian tumorigenesis.

  18. Secretion of protein and epidermal growth factor (EGF) by transplanted human pancreas.

    Science.gov (United States)

    Konturek, J W; Buesing, M; Hopt, U T; Stachura, J; Becker, H D; Konturek, S J

    1992-08-01

    Epidermal growth factor (EGF) has been localized in human salivary and Brunner's glands and found to stimulate the proliferation of gastrointestinal and pancreatic tissues in animals, but little is known about EGF in human pancreas. This study was designed to determine the distribution and release of EGF in the pancreas and to assess the secretion of EGF and protein by the transplanted human pancreas. The peroxidase antiperoxidase (PAP) immunocytochemical method with anti-hEGF showed that EGF was restricted mainly to the excretory cells lining pancreatic ducts. The EGF immunoreactivity in the pancreatic tissue averaged about 15 +/- 0.5 micrograms/g of tissue wt. The concentration and output of EGF in the pancreatic juice were, respectively, about 3.4 +/- 0.7 ng/mL and 68 + 12 ng/h in basal secretion collected from the whole pancreatic transplant. A significant increase in EGF release from this transplant started about 2 h after its reperfusion and was accompanied by a parallel increase in protein output. Injection of iv secretion (1 U/kg) resulted in a transient rise in EGF output, probably as a result of washout by increased vol flow, whereas HCCK (1 U/kg) caused more prolonged release of EGF accompanied by a marked stimulation of protein secretion. Ingestion of a mixed meal caused an immediate and sustained increment in EGF output, and protein output showed a more protracted increase, reaching its peak in the second postprandial hour. Fractionation of an extract of pancreatic juice on G-5O Sephadex superfine column revealed that EGF immunoreactivity emerged as a major peak in the same position as authentic human EGF (hEGF).(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells.

    Science.gov (United States)

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A; Ackerman, Janet M; Yaswen, Paul; Vulpe, Chris D; Leitman, Dale C

    2016-05-01

    Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM, Yaswen P, Vulpe CD, Leitman DC. 2016. Parabens and human epidermal

  20. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells

    Science.gov (United States)

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A.; Ackerman, Janet M.; Yaswen, Paul; Vulpe, Chris D.; Leitman, Dale C.

    2015-01-01

    Background: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. Objectives: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). Methods: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Results: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Conclusion: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Citation: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM

  1. Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

    Science.gov (United States)

    Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

    2016-01-01

    Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

  2. Bone regeneration in experimental animals using calcium phosphate cement combined with platelet growth factors and human growth hormone.

    Science.gov (United States)

    Emilov-Velev, K; Clemente-de-Arriba, C; Alobera-García, M Á; Moreno-Sansalvador, E M; Campo-Loarte, J

    2015-01-01

    Many substances (growth factors and hormones) have osteoinduction properties and when added to some osteoconduction biomaterial they accelerate bone neoformation properties. The materials included 15 New Zealand rabbits, calcium phosphate cement (Calcibon(®)), human growth hormone (GH), and plasma rich in platelets (PRP). Each animal was operated on in both proximal tibias and a critical size bone defect of 6mm of diameter was made. The animals were separated into the following study groups: Control (regeneration only by Calcibon®), PRP (regeneration by Calcibon® and PRP), GH (regeneration by Calcibon® and GH). All the animals were sacrificed at 28 days. An evaluation was made of the appearance of the proximal extreme of rabbit tibiae in all the animals, and to check the filling of the critical size defect. A histological assessment was made of the tissue response, the presence of new bone formation, and the appearance of the biomaterial. Morphometry was performed using the MIP 45 image analyser. ANOVA statistical analysis was performed using the Statgraphics software application. The macroscopic appearance of the critical defect was better in the PRP and the GH group than in the control group. Histologically greater new bone formation was found in the PRP and GH groups. No statistically significant differences were detected in the morphometric study between bone formation observed in the PRP group and the control group. Significant differences in increased bone formation were found in the GH group (p=0.03) compared to the other two groups. GH facilitates bone regeneration in critical defects filled with calcium phosphate cement in the time period studied in New Zealand rabbits. Copyright © 2014 SECOT. Published by Elsevier Espana. All rights reserved.

  3. Transforming Growth Factor-β2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    曹阳; 魏厚仁; 笪邦红; 李忠玉

    2003-01-01

    Whether cultured human trabecular meshwork cells express transforming growth factor-β2 (TGF-β2) messenger RNA (mRNA) and protein was investigated. Total RNA of 106 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β2 messenger RNA, and the PCRproduct was verified by sequencing. Immunohistochemical staining was used to detect TGF-β2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β2 and contribute to the presence of TGF-β2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further in vestigation.

  4. Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF gene.

    Directory of Open Access Journals (Sweden)

    Jenny Leitz

    2014-03-01

    Full Text Available The expression of the human papillomavirus (HPV E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

  5. Oncogenic Human Papillomaviruses Activate the Tumor-Associated Lens Epithelial-Derived Growth Factor (LEDGF) Gene

    Science.gov (United States)

    Leitz, Jenny; Reuschenbach, Miriam; Lohrey, Claudia; Honegger, Anja; Accardi, Rosita; Tommasino, Massimo; Llano, Manuel; von Knebel Doeberitz, Magnus; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2014-01-01

    The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic. PMID:24604027

  6. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    Science.gov (United States)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (Pmuscle cells is likely due to transient membrane disruption on initiation of flow.

  7. Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

    Science.gov (United States)

    Leitz, Jenny; Reuschenbach, Miriam; Lohrey, Claudia; Honegger, Anja; Accardi, Rosita; Tommasino, Massimo; Llano, Manuel; von Knebel Doeberitz, Magnus; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2014-03-01

    The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

  8. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    Science.gov (United States)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (Pmuscle cells is likely due to transient membrane disruption on initiation of flow.

  9. The Effect of Connective Tissue Growth Factor on Human Renal Tubular Epithelial Cell Transdifferentiation

    Institute of Scientific and Technical Information of China (English)

    张春; 朱忠华; 邓安国

    2004-01-01

    To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) . α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).

  10. Nuclear factor-κB mediates placental growth factor induced pro-labour mediators in human placenta.

    Science.gov (United States)

    Lappas, Martha

    2012-07-01

    Prostaglandins, pro-inflammatory cytokines, extracellular matrix remodelling enzymes and nuclear factor-kappa B (NF-κB) are involved in the mechanisms of term and preterm parturition. Recent studies have reported an increase in angiogenesis-related genes during term and preterm labour, including placental growth factor (PLGF). In non-gestational tissues, PLGF induces inflammation via NF-κB. The aim of this study was to determine the effect of PLGF on the gene expression and release of pro-labour mediators in human placenta. Samples were obtained from normal pregnancies at the time of Caesarean section. Human placenta was incubated in the absence (basal control) or presence of a 10 ng/ml PLGF for 24 h. Inflammatory gene expression was analysed by quantitative RT-PCR, concentration of pro-inflammatory cytokines and prostaglandins was quantified by ELISA, and secretory matrix metalloproteinases (MMPs) activity by zymography. NF-κB DNA-binding activity and IκB-α (inhibitor of NF-κB) protein degradation were analysed by ELISA and Western blotting, respectively. PLGF significantly increased interleukin (IL)-6 and IL-8 gene expression and secretion, cyclooxygenase-2 expression and resultant prostaglandin (PG) E(2) and PGF(2α) release, and MMP-9 gene expression and enzyme production. PLGF induced the degradation of IκB-α whilst increasing NF-κB p65 DNA-binding activity. The PLGF-induced pro-labour responses were abrogated by co-treatment with the NF-κB inhibitor BAY 11-7082. In summary, the pro-inflammatory and pro-labour effects of PLGF in human placenta are mediated by NF-κB.

  11. Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin.

    Science.gov (United States)

    Deng, Yi-Ting; Chen, Hsin-Ming; Cheng, Shih-Jung; Chiang, Chun-Pin; Kuo, Mark Yen-Ping

    2009-09-01

    Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We immunohistochemically examined the expression of CTGF protein in 20 cases of OSF and found positive CTGF staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed that arecoline, a main alkaloid found in AN, stimulated CTGF synthesis in a dose- and time-dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CTGF during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-kappaB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 and antioxidant N-acetyl-l-cysteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CTGF synthesis. Furthermore, curcumin completely inhibited arecoline-induced CTGF synthesis and the inhibition is dose-dependent. These results indicated that arecoline-induced CTGF synthesis was mediated by ROS, NF-kappaB, JNK, P38 MAPK pathways and curcumin could be a useful agent in controlling OSF.

  12. Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Introduction Nerve growth factor (NGF) was first discovered and purified by Rita Levi-Montalcini and Stanley Cohen in the 1950s[1,2]. It represents the first cellular growth factor ever discovered and involved in the growth, survival, and differentiation of specific nerve cell populations[3]. Although animal tests and phase-Ⅱ clinical trials indicate that rhNGF could be an effective treatment for diabetic[4] and HIV-related neuropathies[5] , a large-scale phase-Ⅲ clinical trial has failed to give similar result[6].

  13. Human blood-brain barrier insulin-like growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Duffy, K.R.; Pardridge, W.M.; Rosenfeld, R.G.

    1988-02-01

    Insulin-like growth factor (IGF)-1 and IGF-2, may be important regulatory molecules in the CNS. Possible origins of IGFs in brain include either de novo synthesis or transport of circulating IGFs from blood into brain via receptor mediated transcytosis mechanisms at the brain capillary endothelial wall, ie, the blood-brain barrier (BBB). In the present studies, isolated human brain capillaries are used as an in vitro model system of the human BBB and the characteristics of IGF-1 or IGF-2 binding to this preparation were assessed. The total binding of IGF-2 at 37 degrees C exceeded 130% per mg protein and was threefold greater than the total binding for IGF-1. However, at 37 degrees C nonsaturable binding equaled total binding, suggesting that endocytosis is rate limiting at physiologic temperatures. Binding studies performed at 4 degrees C slowed endocytosis to a greater extent than membrane binding, and specific binding of either IGF-1 or IGF-2 was detectable. Scatchard plots for either peptide were linear and the molar dissociation constant of IGF-1 and IGF-2 binding was 2.1 +/- 0.4 and 1.1 +/- 0.1 nmol/L, respectively. Superphysiologic concentrations of porcine insulin inhibited the binding of both IGF-1 (ED50 = 2 micrograms/mL) and IGF-2 (ED50 = 0.5 microgram/mL). Affinity cross linking of /sup 125/I-IGF-1, /sup 125/I-IGF-2, and /sup 125/I-insulin to isolated human brain capillaries was performed using disuccinimidylsuberate (DSS). These studies revealed a 141 kd binding site for both IGF-1 and IGF-2, and a 133 kd binding site for insulin.

  14. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.

    Science.gov (United States)

    Babavalian, H; Latifi, A M; Shokrgozar, M A; Bonakdar, S; Tebyanian, H; Shakeri, F

    2016-07-31

    The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

  15. Platelet-derived growth factor and platelet-derived growth factor receptor-α expression in the normal human thymus and thymoma

    Science.gov (United States)

    Cimpean, Anca Maria; Ceauşu, Raluca; Encică, Svetlana; Gaje, Pusa Nela; Ribatti, Domenico; Raica, Marius

    2011-01-01

    Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) are strongly involved in the normal development of several organs, tumour angiogenesis and malignant progression and metastasis. Few studies concerning their expression, distribution and role in normal and pathological human thymus are available in the literature. The aim of this study has been to analyse the immunohistochemical expression of PDGF and PDGFR-α in prenatal and postnatal normal human thymus and thymomal biopsy specimens. The results demonstrated immunoreactivity to both PDGF and PDGFR-α in all specimens, but the intensity, distribution and number of positive cells were different in normal thymus and thymomas, and also among different tumour types. PDGF and PDGFR-α were weakly expressed in foetal and postnatal humans with a different distribution between cortex and medulla in both blood vessels and epithelial cells, whereas they were overexpressed in thymoma, especially in type B2 and B3, in the tumour epithelial cells. Overall, these data suggest that PDGF and PDGFR-α may be involved in the pathophysiology of the human thymus. PMID:21645144

  16. Platelet-derived growth factor and platelet-derived growth factor receptor-α expression in the normal human thymus and thymoma.

    Science.gov (United States)

    Cimpean, Anca Maria; Ceauşu, Raluca; Encică, Svetlana; Gaje, Pusa Nela; Ribatti, Domenico; Raica, Marius

    2011-10-01

    Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) are strongly involved in the normal development of several organs, tumour angiogenesis and malignant progression and metastasis. Few studies concerning their expression, distribution and role in normal and pathological human thymus are available in the literature. The aim of this study has been to analyse the immunohistochemical expression of PDGF and PDGFR-α in prenatal and postnatal normal human thymus and thymomal biopsy specimens. The results demonstrated immunoreactivity to both PDGF and PDGFR-α in all specimens, but the intensity, distribution and number of positive cells were different in normal thymus and thymomas, and also among different tumour types. PDGF and PDGFR-α were weakly expressed in foetal and postnatal humans with a different distribution between cortex and medulla in both blood vessels and epithelial cells, whereas they were overexpressed in thymoma, especially in type B2 and B3, in the tumour epithelial cells. Overall, these data suggest that PDGF and PDGFR-α may be involved in the pathophysiology of the human thymus.

  17. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  18. Quantitative analysis using ELISA of vascular endothelial growth factor and basic fibroblast growth factor in human colorectal cancer, liver metastasis of colorectal cancer and hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Muriel Mathonnet; Bernard Descottes; Denis Valleix; Fran(c)ois Labrousse; Véronique Truffinet; Yves Denizot

    2006-01-01

    @@ TO THE EDITOR Angiogenesis consists of the sprouting of capillaries from pre-existing vessels[1]. It is well-known that tumor growth is angiogenesis-dependent. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)stimulated vascular endothelial cell proliferation and are involved in the neoplastic angiogenesis of several types of tumors including those of the intestinal tract[1-5].

  19. Comparison of the levels of the growth factors in umbilical cord serum and human milk and its clinical significance.

    Science.gov (United States)

    Patki, Satish; Patki, Ujjwala; Patil, Rajendra; Indumathi, S; Kaingade, Pankaj; Bulbule, Akshata; Nikam, Amar; Pishte, Amit

    2012-08-01

    The process of the growth of the fetus begins in the uterus and gets further accelerated following the birth, especially during initial few months. The role of the growth factors in the physiology of the cellular growth is already well established. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) seem to be imperative for angiogenesis, cell development and proliferation as well as maintenance of the tissues. The levels of these factors in the maternal serum during pregnancy as well as during postpartum period are insignificant. Consequently, we hypothesized that the fetus receives moderate supply of these growth factors from the placenta during its stay in the uterus. This supply gets further augmented during the postpartum period through the different source, i.e. mother's milk. To study this physiological transition of the source of the growth factors from the placenta to the breast milk, the concentrations of VEGF and HGF in the cord serum of full term neonates and that in the breast milk of the corresponding mothers were analyzed during ELISA. The human milk, especially the colostrum revealed significantly higher levels of VEGF and HGF (1541.759 ± 119.349 pg/ml and 7129.249 ± 273.472 pg/ml) than cord serum (16.632 ± 0.773 pg/ml and 2581.6 ± 108.275 pg/ml) respectively. The multifold higher levels of VEGF observed in colostrum probably correlates with its high neonatal requirement for the maturation of the gastrointestinal epithelium following birth. The higher levels of both the growth factors in the breast milk than those observed in the cord serum probably explain their higher needs by the neonates for immunological protection, protein synthesis and neurocognitive development. The observations of the present study strengthen the policy of the colostrum feeding, which is promoted by organizations like World Health Organization (WHO). This study further documents the fact that the commercial milk formulae cannot replace the human

  20. Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

    Institute of Scientific and Technical Information of China (English)

    GUO; Jianli; QU; Shen

    2001-01-01

    A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL601 cells and was cloned into a eukaryotic expressing vector pcDNA3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL601 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

  1. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

    Science.gov (United States)

    Vogt, Rhonda R; Unda, Richard; Yeh, Lee-Chuan C; Vidro, Eileen K; Lee, John C; Tsin, Andrew T

    2006-08-01

    Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

  2. Lapatinib plus trastuzumab in pretreated human epidermal growth factor receptor 2-positive metastatic breast cancer

    Directory of Open Access Journals (Sweden)

    Miguel J Sotelo

    2014-01-01

    Full Text Available Background: Dual human epidermal growth factor receptor 2 (HER2 blockade has been preclinically and clinically assessed in HER2-overexpressing metastatic breast cancer (mBC with encouraging results. Patients and Methods: This is a descriptive retrospective study of trastuzumab plus lapatinib activity in patients with HER2-overexpressing mBC from two centers. The primary endpoints were to assess objective response rate (ORR and toxicity. The secondary endpoints were to assess progression-free survival (PFS and overall survival. Results: A total of 23 HER2-positive mBC patients previously treated with trastuzumab received a trastuzumab plus lapatinib based therapy. Chemotherapy (CT was added to the dual HER2 blockade treatment in 13 patients (56%, whereas hormonotherapy (HT was added in 8 patients (35% and 2 patients (9% received lapatinib plus trastuzumab without any other agent. ORR was 22% (5/23 and 39% (9/23 of patients had stable disease. PFS in the overall population was 4 months. PFS in patients with CT was 5 months, whereas PFS in patients with HT was 2 months. Grade ≥ 3 adverse events were diarrhea (26% and hand-and-foot syndrome (9%. Conclusions: These findings suggest that dual HER2 blockade in combination with CT is feasible in pretreated HER2-positive mBC patients.

  3. Structural study of human growth hormone-releasing factor fragment (1?29) by vibrational spectroscopy

    Science.gov (United States)

    Carmona, P.; Molina, M.; Lasagabaster, A.

    1995-05-01

    The conformational structure of fragment 1-29 of human growth hormone releasing factor, hGHRF (1-29), in aqueous solution and in the solid state is investigated by infrared and Raman spectroscopy. The polypeptide backbone is found to be unordered in the solid state. However, the spectra of the peptide prepared as 5% (w/w) aqueous solutions show that approximately 28% of the peptide is involved in intermolecular β-sheet aggregation. The remainder of the peptide exists largely as disordered and β-sheet conformations with a small portion of α-helices. Tyrosine residues are found to be exposed to the solvent. The secondary structures are quantitatively examined through infrared spectroscopy, the conformational percentages being near those obtained by HONDAet al. [ Biopolymers31, 869 (1991)] using circular dichroism. The fast hydrogen/deuterium exchange in peptide groups and the absence of any NMR sign indicative of ordered structure [ G. M. CLOREet al., J. Molec. Biol.191, 553 (1986)] support that the solution conformations of the non-aggregated peptide interconvert in dynamic equilibrium. Some physiological advantages that may derive from this conformational flexibility are also discussed

  4. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Jing; Wang; Ting-Jun; Fan; Xiu-Xia; Yang; Shi-Min; Chang

    2014-01-01

    AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P <0.01) and the length of F-actin,reduced the mean optical density(P <0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  5. Pertuzumab in human epidermal growth-factor receptor 2-positive breast cancer: clinical and economic considerations

    Directory of Open Access Journals (Sweden)

    Lamond NW

    2014-05-01

    Full Text Available Nathan WD Lamond, Tallal YounisDepartment of Medicine, Dalhousie University at the Queen Elizabeth II Health Sciences Centre, Halifax, NS, CanadaAbstract: In the absence of specific therapy, the 15%–20% of breast cancers demonstrating human epidermal growth-factor receptor 2 (HER2 protein overexpression and/or gene amplification are characterized by a more aggressive phenotype and poorer prognosis compared to their HER2-negative counterparts. Trastuzumab (Herceptin, the first anti-HER2-targeted therapy, has been associated with improved survival outcomes in HER2-positive breast cancer. However, many patients with early stage disease continue to relapse, and metastatic disease remains incurable. In order to further improve these outcomes, several novel HER2-targeted agents have recently been developed. Pertuzumab (Perjeta, a monoclonal antibody against the HER2 dimerization domain, has also been associated with improved patient outcomes in clinical trials, and has recently been approved in combination with chemotherapy and trastuzumab for neoadjuvant therapy of early stage, HER2-positive breast cancer and first-line treatment of metastatic disease. This review briefly summarizes pertuzumab's clinical development as well as the published evidence supporting its use, and highlights some of the currently unanswered questions that will influence pertuzumab’s incorporation into clinical practice.Keywords: HER2/neu, clinical trials, drug development, novel therapies, targeted anticancer therapy

  6. Human epidermal growth factor receptor 2 expression in mixed gastric carcinoma.

    Science.gov (United States)

    Wang, Yang-Kun; Chen, Zhong; Yun, Tian; Li, Cong-Yang; Jiang, Bo; Lv, Xue-Xia; Chu, Guang-Hui; Wang, Su-Nan; Yan, Hui; Shi, Lei-Feng

    2015-04-21

    To investigate human epidermal growth factor receptor 2 (HER2) amplification and protein expression in mixed gastric carcinoma. Fluorescence in situ hybridization and immunohistochemistry were used to detect HER2 amplification and protein expression in 277 cases of mixed gastric carcinoma. Protein staining intensity was rate as 1+, 2+, or 3+. Of the 277 cases, 114 (41.2%) expressed HER2 protein. HER2 3+ staining was observed in 28/277 (10.1%) cases, 2+ in 37/277 (13.4%) cases, and 1+ in 49/277 (17.7%) cases. A HER2 amplification rate of 17% was detected, of which 25/28 (89.3%) were observed in the HER2 3+ staining group, 17/37 (45.9%) in 2+, and 5/49 (10.2%) in 1+. Of the 47 patients with HER2 amplification who received chemotherapy plus trastuzumab, 22 demonstrated median progression-free and overall survivals of 9.1 mo and 16.7 mo, respectively, which were significantly better than those achieved with chemotherapy alone (5.6 mo and 12.1 mo, respectively) in 19 previously treated patients (Ps gastric carcinoma displays high heterogeneity. Relatively quantitative parameters are needed for assessing the level of HER2 amplification and protein expression.

  7. Free insulin-like growth factors (IGF-I and IGF-II) in human serum.

    Science.gov (United States)

    Frystyk, J; Skjaerbaek, C; Dinesen, B; Orskov, H

    1994-07-11

    Using ultrafiltration by centrifugation we have isolated the free, unbound fractions of insulin-like growth factor I and II (free IGF-I and IGF-II) in human serum. In this way near in vivo conditions could be maintained before and during isolation. The recovery was 80 to 100% in the ultrafiltrates, which contained no detectable amounts of IGF-binding proteins (IGFBPs) as measured by Western ligand blotting and IGFBP-1 and IGFBP-3 immunoassays. The concentration of free peptides was measured in two ultrasensitive non-competitive IGF-I and IGF-II time-resolved fluoroimmunoassays. We found that (i) equilibrium between free and protein-complexed IGF was strongly dependent on re-establishment of in vivo conditions (temperature, pH, ionic milieu and dilution); (ii) metabolic events (glucose load and fasting) caused significant changes in free IGF-I and IGF-II levels without concomitant changes in total circulating levels of IGFs; (iii) in 49 healthy adult subjects (20 to above 60 years) free IGF-I was inversely related to age and ranged from 950 +/- 150 ng/l (mean +/- S.E.M.) (20-30 years) to 410 +/- 70 ng/l (> 60 years). The relative percentage was, however, unchanged, being 0.38 +/- 0.02% of total IGF-I. In contrast, free IGF-II was independent of age, being 1,480 +/- 80 ng/l (approximately 0.20 +/- 0.01% of total IGF-II).

  8. Expression and purification of human epidermal growth factor (hEGF fused with GB1

    Directory of Open Access Journals (Sweden)

    Xueming Zheng

    2016-07-01

    Full Text Available Fusion expression is a promising strategy for the production of bioactive peptides in Escherichia coli. In this study, we constructed a new recombinant expression plasmid containing the coding sequence of 56-residue B1 domain of streptococcal protein G (GB1. For easy purification and cleavage of the recombinant proteins, except GB1, an engineered hexahistidine and tobacco etch virus (TEV protease recognition sites were included in the fusion sequence. Next, we cloned the coding sequence of human epidermal growth factor (hEGF into this new plasmid and produced the recombinant hEGF in E. coli. The bioactive hEGF is a 53-amino acid peptide and is stabilized by three intramolecular disulphide bonds. Compared with glutathione S-transferase, thioredoxin and small ubiquitin-related modifier, GB1 greatly improved the expression and solubility of hEGF. Moreover, the recombinant hEGF bound to the nickel nitrilotriacetic acid resin column, was easily cleaved by TEV protease and the free hEGF was released. The results showed that this new plasmid was appropriate for recombinant production of small bioactive peptides, such as hEGF, which contains a high proportion of hydrophobic residues and intramolecular disulphide linkages.

  9. Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment

    Energy Technology Data Exchange (ETDEWEB)

    Koo, Tae Ryool [Dept. of Radiation Oncology, Hallym University Chuncheon Sacred Heart Hospital, Chuncheon (Korea, Republic of); Kim, In Ah [Dept. of Radiation Oncology, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of)

    2016-03-15

    Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents.

  10. Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes.

    Science.gov (United States)

    Turpaev, K; Litvinov, D; Dubovaya, V; Panasyuk, A; Ivanov, D; Prassolov, V

    2001-06-01

    We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

  11. Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

    Science.gov (United States)

    Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

    2013-11-07

    The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

  12. EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    司晓辉; 刘正

    2001-01-01

    Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts ( HPDLFs ). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-β and rhBMP2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin ( OC) synthesis and formation of the mineralized nodules, respectively. Results TGF-β(5~100ng /ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng /ml TGF-β. TGF-β(0.5~100ng /ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0.25~2mg/ ml) had no rernarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and formation of the mineralized nodules of HPDLFs were significantly stimulated by 0.5~2mg/ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-β can stimulate HPDLFs to express the early marker of osteoblastic phenotype , and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblastic phenotype of HPDLFs.

  13. Transforming growth factor-beta inhibits human antigen-specific CD4(+) T cell proliferation without modulating the cytokine response

    NARCIS (Netherlands)

    Tiemessen, MM; Kunzmann, S; Schmidt-Weber, CB; Garssen, J; Bruijnzeel-Koomen, CAFM; Knol, EF; Van Hoffen, E

    2003-01-01

    Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated y

  14. Using growth factors in human extraction sockets: a histologic and histomorphometric evaluation of short-term healing

    NARCIS (Netherlands)

    Geurs, N.; Ntounis, A.; Vassilopoulos, P.; van der Velden, U.; Loos, B.G.; Reddy, M.

    2014-01-01

    Purpose: Ridge preservation protocols reduce crestal remodeling after tooth extraction. There is insufficient evidence on bone grafting in combination with platelet-rich plasma (PRP) or recombinant human platelet-derived growth factor (rhPDGF-BB). The aim of this study is to evaluate healing of graf

  15. Vascular endothelial growth factor receptor-1 contributes to resistance to anti-epidermal growth factor receptor drugs in human cancer cells.

    Science.gov (United States)

    Bianco, Roberto; Rosa, Roberta; Damiano, Vincenzo; Daniele, Gennaro; Gelardi, Teresa; Garofalo, Sonia; Tarallo, Valeria; De Falco, Sandro; Melisi, Davide; Benelli, Roberto; Albini, Adriana; Ryan, Anderson; Ciardiello, Fortunato; Tortora, Giampaolo

    2008-08-15

    The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

  16. Binding of insulin-like growth factors to Tera-2 human embryonal carcinoma cells during differentiation.

    Science.gov (United States)

    Fleck, J F; Sledge, G W; Benenati, S V; Frolik, C A; Roth, B J; Hirsch, K S

    1991-08-15

    Differentiation of Tera-2 human embryonal carcinoma cells by exposure to 2.1 mM alpha-difluoromethylornithine resulted in changes in morphology, a decrease in growth rate, and changes in the expression of SSEA-1 differentiation antigen. While the binding of 125I-insulin-like growth factor I (IGF-I) remained relatively constant during differentiation, binding of 125I-IGF-II increased 2-3-fold. Further, the binding of IGF-II was 87 times greater than IGF-I in both undifferentiated and differentiated cells. Undifferentiated Tera-2 cells exhibited a single class of binding sites for both IGF-I (KD = 1.2 nM, 7.0 x 10(3) sites/cell) and IGF-II (KD = 8.3 nM, 3.4 x 10(5) sites/cell). Following differentiation, IGF-I continued to bind to a single class of binding sites (KD 1.0 nM, 6.7 x 10(3) sites/cell) whereas IGF-II bound to both high-affinity sites (KDH 0.3 nM, 2.2 x 10(5) sites/cell) and low-affinity sites (KDL 15.1 nM, 1.6 x 10(7) sites/cell). The binding of iodinated IGF-II was blocked by unlabeled IGF-II but not IGF-I. In contrast, 125I-IGF-I binding was prevented by either IGF-I or IGF-II. Affinity cross-linking experiments demonstrated the presence of both type I and type II IGF receptors along with a number of IGF binding proteins. IGF-I failed to stimulate the incorporation of [3H]thymidine in both undifferentiated and differentiated cells. Although IGF-II caused a significant increase in [3H]thymidine incorporation in both undifferentiated and differentiated Tera-2 cells, the magnitude of the response and the sensitivity of the cells to IGF-II stimulation was diminished following differentiation. The observed changes in IGF-II binding, which occur in conjunction with cellular differentiation, may be an important feature of the expression of the differentiated phenotype by human germ cell tumors.

  17. Tissue engineering of urethra using human vascular endothelial growth factor gene-modified bladder urothelial cells.

    Science.gov (United States)

    Guan, Yong; Ou, Lailiang; Hu, Gang; Wang, Hongjun; Xu, Yong; Chen, Jiatong; Zhang, Jun; Yu, Yaoting; Kong, Deling

    2008-02-01

    Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF(165)) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF(165)-GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF(165)-GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF(165) was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the

  18. Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods.

    Science.gov (United States)

    Kim, Nam Ah; Lim, Dae Gon; Lim, Jun Yeul; Kim, Ki Hyun; Jeong, Seong Hoon

    2015-02-01

    Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.

  19. Human hepatocyte growth factor (hHGF-modified hepatic oval cells improve liver transplant survival.

    Directory of Open Access Journals (Sweden)

    Zhu Li

    Full Text Available Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF in modifying hepatic oval cells (HOCs administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05. Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2, tumor necrosis factor-α (TNF-α and interferon-γ (IFN-γ levels while increasing the production of IL-10 and TGF-β1 (P<0.05. HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only. Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver

  20. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection.

    OpenAIRE

    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...

  1. Interleukin 15 is a growth factor for human thymocytes with preferential effect on CD8(+)cells

    DEFF Research Database (Denmark)

    Thulesen, S; Nielsen, M; Petersen, T R

    2000-01-01

    The effects of IL-15, as compared to IL-2, on growth of human thymocytes has been evaluated. Expression of comparable amounts of receptor chains was found on IL-2 and IL-15 cultured thymocytes, as well as comparable receptor signalling. However, IL-15 was superior to IL-2 in promoting CD8(+)thymo...

  2. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  3. HUMAN CAPITAL OF THE REGIONS-DRIVEN FACTOR FOR GROWTH AND EMPLOYMENT.THE CASE OF ROMANIA

    Directory of Open Access Journals (Sweden)

    Olimpia Neagu

    2011-01-01

    Full Text Available The world is entering a new era - a time when economic wealth is created by knowledgeand skills and the human capital of nations, regions and cities will determine the winnersfrom the loosers. The paper is focused on the human capital’s performance of the romanianregions, measured by economic growth and employment, analysing statistical data fromeuropean sources. There are differences between regions regarding the human capitalendowment, employment and economic growth. The assumption of a positive correlationbetween these variables is partially confirmed. Further researches are needed to measure theimpact of other factors such as: human migration or attractivity of regions. Designing anddeveloping regional human capital strategies would be a good starting point for a positiveperspective of the Europe 2020 strategy implementation.

  4. Molecular level interaction of the human acidic fibroblast growth factor with the antiangiogenic agent, inositol hexaphosphate .

    Science.gov (United States)

    Kumar, Sriramoju M; Wang, Han-Min; Mohan, Sepuru K; Chou, Ruey-Hwang; Yu, Chin

    2010-12-21

    Acidic fibroblast growth factor (FGF1) regulates a wide array of important biological phenomena such as angiogenesis, cell differentiation, tumor growth, and neurogenesis. Generally, FGFs are known for their strong affinity for the glycosaminoglycan heparin, as a prerequisite for recognition of a specific tyrosine kinase on the cell surface and are responsible for the cell signal transduction cascade. Inositol hexaphosphate (IP6) is a natural antioxidant and is known for its antiangiogenic role, in addition to its ability to control tumor growth. In the present study, we investigated the interaction of IP6 with the acidic fibroblast growth factor (FGF1) using various biophysical techniques including isothermal calorimetry, circular dichroism, and multidimensional NMR spectroscopy. Herein, we have reported the three-dimensional solution structure of the FGF1-IP6 complex. These data show that IP6 binds FGF1 and enhances its thermal stability. In addition, we also demonstrate that IP6 acts as an antagonist to acidic fibroblast growth factor by inhibiting its receptor binding and subsequently decreasing the mitogenic activity. The inhibition likely results in the ability of IP6 to antagonize the angiogenic and mitogenic activity of FGF1.

  5. Structural alterations of transforming growth factor-beta receptor genes in human cervical carcinoma

    NARCIS (Netherlands)

    Chen, TP; De Vries, EGE; Hollema, H; Yegen, HA; Vellucci, VF; Strickler, HD; Hildesheim, A; Reiss, M

    1999-01-01

    The development and progression of invasive uterine cervical carcinomas appear to be associated with the progressive loss of sensitivity to transforming growth factor-beta (TGF beta)-mediated cell cycle arrest. In order to identify possible molecular mechanisms responsible for TGF beta resistance, w

  6. Epidermal growth factor inhibits glycyl sarcosine transport and hPepT1 expression in a human intestinal cell line

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Amstrup, Jan; Steffansen, Bente;

    2001-01-01

    Intestinal oligopeptide transporter, growth factor, immunocytochemistry, laser scanning confocal microscopy......Intestinal oligopeptide transporter, growth factor, immunocytochemistry, laser scanning confocal microscopy...

  7. Solution structure of human insulin-like growth factor II; recognition sites for receptors and binding proteins.

    OpenAIRE

    Terasawa, H; Kohda, D.; Hatanaka, H; Nagata, K.; Higashihashi, N; Fujiwara, H.; Sakano, K; Inagaki, F.

    1994-01-01

    The three-dimensional structure of human insulin-like growth factor II was determined at high resolution in aqueous solution by NMR and simulated annealing based calculations. The structure is quite similar to those of insulin and insulin-like growth factor I, which consists of an alpha-helix followed by a turn and a strand in the B-region and two antiparallel alpha-helices in the A-region. However, the regions of Ala1-Glu6, Pro31-Arg40 and Thr62-Glu67 are not well-defined for lack of distanc...

  8. Expression of Human Epidermal Growth Factor Receptor-2 in Resected Rectal Cancer

    Science.gov (United States)

    Meng, Xiangjiao; Huang, Zhaoqin; Di, Jian; Mu, Dianbin; Wang, Yawei; Zhao, Xianguang; Zhao, Hanxi; Zhu, Wanqi; Li, Xiaolin; Kong, Lingling; Xing, Ligang

    2015-01-01

    Abstract The addition of trastuzumab to chemotherapy was demonstrated to be beneficial for advanced human epidermal growth factor receptor-2 (HER-2) positive gastric cancer. However, the HER-2 status of rectal cancer remains uncertain. This study aimed to determine the HER-2 expression in a large multicenter cohort of rectal cancer patients. The clinical and pathological features of 717 patients were retrospectively reviewed. All the patients were diagnosed with primary rectal adenocarcinoma without distant metastasis and took surgery directly without any preoperative anticancer treatment. HER-2 status was assessed on resected samples. A total of 99 cases with IHC3+ and 16 cases with IHC 2+ plus gene amplification were determined as HER-2 positive. 22.6% of HER-2 positive patients had local recurrence, whereas 16.9% of HER-2 negative patients did (P = 0.146). HER-2 positive tumors were more likely to have distant metastasis (P = 0.007). Univariate analysis revealed that pathological tumor stage, pathological node stage, positive margin, and lymphovascular invasion were significantly correlated with 5-year disease-free survival (DFS) and 5-year overall survival (OS). The patients with >10 dissected lymph nodes showed significantly longer OS (P = 0.045) but not DFS (P = 0.054). HER-2 negative patients had significantly better 5-year DFS (P < 0.001) and 5-year OS (P = 0.013) than those of the HER-2 positive patients. In the subgroup analysis for the early rectal cancer and locally advanced rectal cancer, HER-2 was also a poor predictor for survival. Multivariate analysis revealed that HER-2 was an independent prognostic factor for 5-year DFS (hazard ratio [HR] = 1.919, 95% confidence interval [CI] 1.415–2.605, P < 0.001) and for 5-year OS (HR = 1.549, 95% CI 1.097–2.186, P = 0.013). When the treatment was included in the analysis for locally advanced patients, HER-2 was a prognostic factor for 5-year DFS (P = 0.001) but not for

  9. Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Orcel, P.; Bielakoff, J.; De Vernejoul, M.C. (INSERM U18, Hopital Lariboisiere, Paris (France))

    1990-02-01

    Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes.

  10. A macroporous bioreactor super activated by the recombinant human transforming growth factor-beta 3

    Directory of Open Access Journals (Sweden)

    Ugo eRipamonti

    2012-06-01

    Full Text Available Macroporous single-phase hydroxyapatite (HA and biphasic HA/β-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/β-TCP were combined with 25 or 125 µg recombinant human transforming growth factor-β3 (hTGF-β3 to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100% and partially converted to 5% and 13% hydroxyapatite/calcium carbonate (HA/CC preloaded with 125 and 250 µg hTGF-β3, and 1:5 and 5:1 binary applications of hTGF-β3: hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-β3. Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly-formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 % and 13 % HA/CC pre-loaded with 125 and 250 µg hTGF-β3 showed substantial induction of bone formation; 250 µg hTGF-β3 induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e. the 1:5 hTGF-β3:hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 µg hTGF-β3 resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

  11. First in human nanotechnology doxorubicin delivery system to target epidermal growth factor receptors in recurrent glioblastoma.

    Science.gov (United States)

    Whittle, James R; Lickliter, Jason D; Gan, Hui K; Scott, Andrew M; Simes, John; Solomon, Benjamin J; MacDiarmid, Jennifer A; Brahmbhatt, Himanshu; Rosenthal, Mark A

    2015-12-01

    There are limited treatment options for patients with recurrent glioblastoma (GBM). The EnGeneIC delivery vehicle (EDV) is a novel nanocellular (minicell) compound which packages theoretically effective concentrations of chemotherapeutic drugs that are designed to target tumors via minicell-surface attached bispecific proteins (EnGeneIC, Lane Cove West, NSW, Australia). Epidermal growth factor receptor (EGFR) is overexpressed in 40-50% of patients with GBM and is a promising target for new therapeutics. (V)EDVDox contains doxorubicin (Dox) within the minicells and targets EGFR through Vectibix (V; Amgen Biologicals, Thousand Oaks, CA, USA). We conducted a first in human Phase I study of (V)EDVDox in adults with recurrent GBM expressing EGFR on immunohistochemistry, following standard therapy including radiation and temozolomide, to establish a safe maximum tolerated dose and determine a recommended Phase II dose (RPTD). (V)EDVDox was administered weekly in an 8week cycle, with dose escalation in successive cohorts of patients using a standard 3+3 design. In total, 14 patients were treated at three dose levels, and the RPTD was identified as 5×10(9)(V)EDVDox. Overall (V)EDVDox was well tolerated, with no dose limiting toxicity and no withdrawals from the study due to adverse events. The most common adverse events were nausea, fever, and chills or rigors, experienced in seven, five and five patients, respectively. Transient uncomplicated hypophosphatemia was seen in seven patients and was not dose-related. Our results demonstrate that (V)EDVDox, up to a dose of 5×10(9)(V)EDVDox weekly, is well tolerated in patients with recurrent GBM.

  12. Human epidermal growth factor receptor 2/neu protein expression in meningiomas: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Ramesh Babu Telugu

    2016-01-01

    Full Text Available Background: Meningiomas are common slow-growing primary central nervous system tumors that arise from the meningothelial cells of the arachnoid and spinal cord. Human epidermal growth factor receptor 2 (HER2 or HER2/neu (also known as c-erbB2 is a 185-kD transmembrane glycoprotein with tyrosine kinase activity expressed in meningiomas and various other tumors. It can be used in targeted therapy for HER2/neu positive meningiomas. Aim: To correlate the expression of HER2/neu protein in meningiomas with gender, location, histological subtypes, and grade. Materials and Methods: It was 3½ years prospective (March 2010–October 2011 and retrospective (May 2008–February 2010 study of histopathologically diagnosed intracranial and intraspinal meningiomas. Clinical details of all the cases were noted from the computerized hospital information system. Immunohistochemistry for HER2/neu protein was performed along with scoring. Statistical analysis was done using Chi-square test to look for any association of HER2/neu with gender, location, grade, and various histological subtypes of meningiomas at 5% level of significance. Results: A total of 100 cases of meningiomas were found during the study period. Of which, 80 were Grade I, 18 were Grade II, and 2 were Grade III meningiomas as per the World Health Organization 2007 criteria. The female-male ratio was 1.9:1 and the mean age was 47.8 years. HER2/neu protein was expressed in 75% of Grade I and 72.2% of Grade II and none of Grade III meningiomas. About 72.7% brain invasive meningiomas showed HER2/neu immunopositivity. Conclusion: HER2/neu protein was expressed in 73% of meningiomas. Statistically significant difference of HER2/neu expression was not seen between females and males of Grade I and Grade II/III meningiomas, intracranial and spinal tumors, Grade I and Grade II/III cases, and various histological subtypes of meningiomas.

  13. Conformational origin of a difficult coupling in a human growth hormone releasing factor analog.

    Science.gov (United States)

    Deber, C M; Lutek, M K; Heimer, E P; Felix, A M

    1989-01-01

    During the solid-phase synthesis of the human growth hormone releasing factor (GRF) analog [Ala15, Leu27, Asn28] -GRF(1-32)-OH, incorporation of Boc-Gln16 was determined to be incomplete. While aggregation of growing resin-bound peptide chains with concomitant beta-sheet formation and "precipitation" has been proposed to account in general for such "difficult coupling," no feature of sequence in the Gln16 region of this GRF analog provided an immediate rationale for this result. We now report 500 MHz 1H NMR spectra of a series of resin-bound GRF segments surrounding the Gln16 position (19-32 through 14-32), swelled in dimethylsulfoxide-d6 solutions [GRF(14-32) = Leu14-Ala-Gln-Leu-Ser(Bzl)-Ala-Arg(Tos)-Lys(CIZ)-Leu- Leu-Gln-Asp(OcHex)-Ile-Leu-Asn-Arg(Tos)-Gln-Gln-Gly32-PAM resin]. While relatively sharp spectra are observed for GRF(19-32), components with resonances broadened by an order-of-magnitude appear in spectra of the 18-32 and 17-32 peptide-resin, and the entire spectrum of 16-32 is ill-resolved and highly broadened. Subsequent spectra sharpen again (15-32, 14-32). These combined synthesis/spectroscopic experimental results, in conjunction with predictive analyses using standard Chou-Fasman 2 degrees structure parameters, suggest that the completeness of the Gln16 coupling is hindered by formation of a specific, folded beta-sheet/beta-turn structure in GRF(16-32) (with the turn located at 18-21, "upstream" of the difficult coupling site), and accompanying aggregation of peptide chains. This analysis suggests that awareness of such potential beta-sheet/beta-turn sequences can guide analog choices, and/or facilitate pre-programming of synthesis steps in anticipation of problem couplings.

  14. Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin

    Institute of Scientific and Technical Information of China (English)

    CHEN Wei; FU Xiao-bing; GE Shi-li; SUN Tong-zhu; SHENG Zhi-yong

    2005-01-01

    Objective : To investigate the expression characteristics of basic fibroblast growth factor (bFGF)and its receptors, flg ( FGFR1 ) and bek ( FGFR2), in fetal skin at different gestational ages underlying the relevance of these 3 proteins to skin development and the mechanisms underlying the phenotypic transition from scarless to scarforming healing.Methods: Eighteen specimens of fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages of 13-32 weeks. Gene expression of bFGF, bek and flg was examined with reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression and distribution of these 3 proteins were detected with streptavidin peroxidase ( SP )immunohistochemical staining method.Results: In the early gestational fetal skin, genes of bFGF and flg were strongly expressed and more protein contents of these 2 proteins were found as compared with the genes at late gestation fetal skin (2.446 ± 0.116 and 2.066 ± 0. 152 versus 2.157 ± 0. 101 and 1.818 ± 0.086,respectively, P < 0.05). On the contrary, the levels of gene expression and protein content of bek were not differently expressed in the early gestational fetal skin versus the late ones. Protein particles of bFGF were mainly distributed in the epidermal cells and some fibroblasts. Bek was mainly located in the cell membrane and cytoplasm of epidermal cells while flg protein was principally located in the epidermal cells, endothelial cells and some fibroblasts.Conclusions: The endogenous bFGF and their receptors might be involved in the cutaneous development at fetal stage. The differently expressing levels of bFGF and flg during gestation may be related to scarless or scarforming repair during gestation.

  15. Freezing adversely affects measurement of vascular endothelial growth factor levels in human aqueous samples

    Directory of Open Access Journals (Sweden)

    Sankarathi Balaiya

    2011-01-01

    Full Text Available Sankarathi Balaiya Sandeep Grover Ravi K Murthy Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Aqueous levels of vascular endothelial growth factor (VEGF can be a surrogate marker of intraocular VEGF activity and a measure of efficacy of anti-VEGF treatment in a variety of vasoproliferative retinal disorders, including diabetic retinopathy, age-related macular degeneration, and central retinal vein occlusion. Measurement of the VEGF level may be adversely affected by premeasurement variables, such as freezing and delay, in sample analysis. We aim to evaluate the effect of storage and delayed measurement of human aqueous VEGF levels in these conditions.Methods: Aqueous samples collected from patients receiving intravitreal injection of bevacizumab for various retinal diseases were divided into two groups. In Group 1, the VEGF levels were analyzed on the same day; in Group 2, the VEGF levels were analyzed after 21 days of freezer storage (-80°C using immunobead assay. Statistical comparison using a paired t-test was performed between the two groups.Results: Thirty-one aqueous humor samples were collected, and the VEGF concentration for fresh samples was 7.8 ± 5.9 pg/mL (mean ± SD compared to 6.5 ± 6.0 pg/mL in frozen samples, resulting in a statistically significant difference (P = 0.03.Conclusions: Accurate measurement of the VEGF level is a vital component of clinical decision-making. Delayed analysis of VEGF levels in aqueous samples may result in significant sample degradation and lower levels of measured VEGF.Keywords: VEGF level, aqueous humor, immunobead assay, VEGF storage

  16. The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

    Directory of Open Access Journals (Sweden)

    Andreas Bayer

    2017-01-01

    Full Text Available Platelet-released growth factors (PRGF and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF® contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3 is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR. In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.

  17. Growth factors and feeder cells promote differentiation of human embryonic stem cell into dopaminergic neurons: a novel role of fibroblast growth factor-20

    Directory of Open Access Journals (Sweden)

    Ana Sofia Correia

    2008-07-01

    Full Text Available Human embryonic stem cells (hESCs are a potential source of dopaminergic neurons for treatment of Parkinson’s disease (PD. Dopaminergic neurons can be derived from hESCs and display a characteristic midbrain phenotype. Once transplanted, they can induce partial behavioral recovery in animal models of PD. The potential research field faces several challenges that need to be overcome before clinical application of hESCs in a transplantation therapy in PD can be considered. These include low survival of the hESC-derived, grafted dopaminergic neurons after transplantation; unclear functional integration of the grafted neurons in the host brain; and, the risk of teratoma/tumor formation from the transplanted cells. This review is focused on our recent efforts to improve the survival of hESC-dervied dopaminergic neurons. We have examined the effect of fibroblast growth factor (FGF-20 in the differentiation of hESCs into dopaminergic neurons. We supplemented cultures of hESCs with FGF-20 during differentiation on PA6 mouse stromal cells for three weeks. When we added FGF-20 the yield of neurons expressing tyrosine hydroxylase increased. We demonstrated that at least part of the effect is contributed by enhanced cell differentiation towards the dopaminergic phenotype as well as reduced cell death. We compare our results with those obtained in other published protocols using different sets of growth factors. Our data indicate that FGF-20 has potent effects to generate large number of dopaminergic neurons derived from hESCs, which may be useful for cell therapy in PD.

  18. Tumour necrosis factor-alpha (TNFα stimulates the growth of human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    F. Rougier

    1997-01-01

    Full Text Available This study reports that TNF-α is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [3H]-thymidine incorporation into DNA and cell counts. In contrast, cytokines such as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-α on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis.

  19. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    Science.gov (United States)

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  20. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    Directory of Open Access Journals (Sweden)

    Acuña-Macías I

    2015-10-01

    Full Text Available Isabel Acuña-Macías,1 Eunice Vera,1 Alma Yolanda Vázquez-Sánchez,1 María Eugenia Mendoza-Garrido,2 Javier Camacho1 1Department of Pharmacology, 2Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico Abstract: Oncogenic ether à-go-go-1 (Eag1 potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. Keywords: lung cancer, serum deprivation, ether à-go-go, potassium channels, EGF, epidermal growth factor, ERK 1/2

  1. Expression of T-Lymphocyte Markers in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer

    Science.gov (United States)

    Lee, Changro; Kim, Joo Heung; Lim, Sung Mook; Park, Hyung Seok; Kim, Seung Il; Park, Byeong-Woo

    2016-01-01

    Purpose The present study aimed to examine the clinical implications of CD4, CD8, and FOXP3 expression on the prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer using a web-based database, and to compare the immunohistochemical expression of T-lymphocyte markers using primary and metastatic HER2-positive tumor tissues before and after HER2-targeted therapy. Methods Using the cBioPortal for Cancer Genomics and Kaplan-Meier plotter, the mRNA expression, association between T-lymphocyte markers, and survival in HER2-positive cancers were investigated according to various cutoff levels. Immunohistochemistry analysis was performed using paired primary and metastatic tissues of 29 HER2-positive tumors treated with systemic chemotherapy and HER2-directed therapy. Results HER2 mRNA was mutually exclusive of T-lymphocyte markers, and a significant correlation between T-cell markers was observed in the cBioPortal for Cancer Genomics. According to analysis of the Kaplan-Meier plotter, the impact of T-lymphocyte marker expression on survival was statistically insignificant in clinical HER2-positive tumors, irrespective of the cutoff levels. However, in the intrinsic HER2-positive subtype, the individual analyses of T-cell markers except for FOXP3 and combined analysis showed significantly favorable survival irrespective of cutoff points. Although the small clinical sample size made it difficult to show the statistical relevance of immunohistochemistry findings, good responses to neoadjuvant treatments might be associated with positive expression of combined T-lymphocyte markers, and approximately half of the samples showed discordance of combined markers between baseline and resistant tumors. Conclusion T-lymphocyte markers could be favorable prognostic factors in HER2-positive breast cancers; however, a consensus on patient section criteria, detection methods, and cutoff value could not be reached. The resistance to HER2-directed therapy might

  2. Human Adenomyosis Endometrium Stromal Cells Secreting More Nerve Growth Factor: Impact and Effect.

    Science.gov (United States)

    Li, Yan; Zou, Shien; Xia, Xian; Zhang, Shaofen

    2015-09-01

    Abnormal expression of nerve growth factor (NGF) was found in adenomyosis (AM). We collected AM foci from patients and eutopic endometrium from non-AM controls. Endometrium stromal cells (ESCs) were cultured. Different levels of 17β-estradiol, tumor necrosis factor (TNF), CoCl2, and H2O2 were added to the culture system separately, then the expression level of NGF in ESCs was detected. After adding different levels of NGF, the proliferation and apoptosis of ESCs and aromatase expression were detected. We found that 17β-estradiol promoted NGF production in AM ESCs but not in control ESCs; TNF promoted NGF production in both AM and control ESCs; and CoCl2 inhibited NGF production in control ESCs, but had no effect in AM ESCs. Nerve growth factor promoted the proliferation and synthesis of aromatase in AM ESCs. In conclusion, locally increased estrogen levels and inflammation may cause increased NGF production in the uterus of patients with AM. Nerve growth factor stimulated the proliferation and increased aromatase expression of ESCs from AM foci, suggesting NGF might contribute to the pathology and etiology of AM.

  3. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

    OpenAIRE

    Fang Zeng; Zicong Li; Qingchun Zhu; Rui Dong; Chengcheng Zhao; Guoling Li; Guo Li; Wenchao Gao; Gelong Jiang; Enqin Zheng; Gengyuan Cai; Stefan Moisyadi; Johann Urschitz; Huaqiang Yang; Dewu Liu

    2017-01-01

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μ...

  4. Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct

    Directory of Open Access Journals (Sweden)

    Erh-Hsuin Lim

    2013-11-01

    Full Text Available BackgroundTo overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF into an electrospun poly(L-lactide scaffold.MethodsThe electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats.ResultsChemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen.ConclusionsWe have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

  5. Transforming growth factor β signaling upregulates the expression of human GDP-fucose transporter by activating transcription factor Sp1.

    Science.gov (United States)

    Xu, Yu-Xin; Ma, Anna; Liu, Li

    2013-01-01

    GDP-fucose transporter plays a crucial role in fucosylation of glycoproteins by providing activated fucose donor, GDP-fucose, for fucosyltransferases in the lumen of the Golgi apparatus. Fucose-containing glycans are involved in many biological processes, which are essential for growth and development. Mutations in the GDP-fucose transporter gene cause leukocyte adhesion deficiency syndrome II, a disease characterized by slow growth, mental retardation and immunodeficiency. However, no information is available regarding its transcriptional regulation. Here, by using human cells, we show that TGF-β1 specifically induces the GDP-fucose transporter expression, but not other transporters tested such as CMP-sialic acid transporter, suggesting a diversity of regulatory pathways for the expression of these transporters. The regulatory elements that are responsive to the TGF-β1 stimulation are present in the region between bp -330 and -268 in the GDP-fucose transporter promoter. We found that this region contains two identical octamer GC-rich motifs (GGGGCGTG) that were demonstrated to be essential for the transporter expression. We also show that the transcription factor Sp1 specifically binds to the GC-rich motifs in vitro and Sp1 coupled with phospho-Smad2 is associated with the promoter region covering the Sp1-binding motifs in vivo using chromatin immunoprecipitation (ChIP) assays. In addition, we further confirmed that Sp1 is essential for the GDP-fucose transporter expression stimulated by TGF-β1 using a luciferase reporter system. These results highlight the role of TGF-β signaling in regulation of the GDP-fucose transporter expression via activating Sp1. This is the first transcriptional study for any nucleotide sugar transporters that have been identified so far. Notably, TGF-β1 receptor itself is known to be modified by fucosylation. Given the essential role of GDP-fucose transporter in fucosylation, the finding that TGF-β1 stimulates the expression of

  6. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Xin-Qin Kang; Wei-Jin Zang; Li-Jun Bao; Dong-Ling Li; Tu-Sheng Song; Xiao-Li Xu; Xiao-Jiang Yu

    2005-01-01

    AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.METHODS: vSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P<0.01). Urea(4.72±1.03 μmol/L) was detected on d 20 (t = 4.272,P<0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCBderived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

  7. Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of vascular endothelial growth factor and connective tissue growth factor in cultured human peritoneal mesothelial cells

    Institute of Scientific and Technical Information of China (English)

    LIU Fu-you; FU Xiao; ZHU Jian-lian; XIAO Li; PENG You-ming; DUAN Shao-bin; LIU Hong; LIU Ying-hong; LING Gui-hui; YUAN Fang; CHEN Jun-xiang

    2007-01-01

    Background The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor β1(TGF-β1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF),a downstream mediator of TGF-β1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells.Methods Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-β1 5 ng/ml, low glucose DMEM + TGF-β1 5 ng/ml + PRS-CTGF-siRNA1-4 and low glucose DMEM + TGF-β1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot.Results Low levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-β1, the levels of CTGF and VEGF were significantly upregulated (P<0.01). Introduction of PRS-CTGF-siRNA1-4 resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P<0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P>0.05).Conclusions The expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-β1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.

  8. Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor

    DEFF Research Database (Denmark)

    Stefaneanu, L; Kovacs, K; Horvath, E

    1989-01-01

    The effect of protracted GH-releasing factor (GRF) stimulation on adenohypophysial morphology was investigated in six mice transgenic for human GRF (hGRF). All animals had significantly higher plasma levels of GH and GRF and greater body weights than controls. Eight-month-old mice were killed...

  9. Adult human dental pulp stem cells promote blood-brain barrier permeability through vascular endothelial growth factor-a expression.

    Science.gov (United States)

    Winderlich, Joshua N; Kremer, Karlea L; Koblar, Simon A

    2016-06-01

    Stem cell therapy is a promising new treatment option for stroke. Intravascular administration of stem cells is a valid approach as stem cells have been shown to transmigrate the blood-brain barrier. The mechanism that causes this effect has not yet been elucidated. We hypothesized that stem cells would mediate localized discontinuities in the blood-brain barrier, which would allow passage into the brain parenchyma. Here, we demonstrate that adult human dental pulp stem cells express a soluble factor that increases permeability across an in vitro model of the blood-brain barrier. This effect was shown to be the result of vascular endothelial growth factor-a. The effect could be amplified by exposing dental pulp stem cell to stromal-derived factor 1, which stimulates vascular endothelial growth factor-a expression. These findings support the use of dental pulp stem cell in therapy for stroke. © The Author(s) 2015.

  10. Choline Phospholipid Metabolites of Human Vascular Endothelial Cells Altered by Cyclooxygenase Inhibition, Growth Factor Depletion, and Paracrine Factors Secreted by Cancer Cells

    Directory of Open Access Journals (Sweden)

    Noriko Mori

    2003-04-01

    Full Text Available Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO, reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs. We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.

  11. Expression of vascular endothelial growth factor and its two receptors in normal human endometrium

    Institute of Scientific and Technical Information of China (English)

    王海燕; 陈贵安

    2003-01-01

    Objectives: We try to demonstrate the expression of vascular endothelial growthfactor (VEGF) and its receptors, flt-1 and KDR, in normal human emdometrium duringthe menstrual cycle.Methods: Immunohistochemical method was used to observe the expression ofVEGF and its two receptors in emdometrium throughout the normal menstrual cyclemeanwhile the isoforms of VEGF were also detected by Western blot analysis. The en-dothelial cells of micro-vessels were marked with Ⅷ factor antibody.Results: VEGF and its receptors existed in endometrial glandular, stromal and vas-cular endothelial cells of human endometrium. Their expressions were higher in the mid-secretory phase of menstrual cycle and highest at menstruation. VEGF121 and VEGF165were the predominant isoforms in normal human endometrium.Conclusion: The expression of VEGF and its two receptors showed cycle-dependentin human endometrium, probably involved in embryonic implantation and endometrialproliferation and differentiation.

  12. Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Han, Bin [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Fujimoto, Naohiro; Matsumoto, Tetsuro [Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Wu, Bin [Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Tanimoto, Akihide [Department of Pathology, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima (Japan); Sasaguri, Yasuyuki [Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Kohno, Kimitoshi, E-mail: k-kohno@med.uoeh-u.ac.jp [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan)

    2011-04-29

    Highlights: {yields} Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. {yields} mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. {yields} Overexpression of mtTFA enhances the growth of cancer cells, whereas downregulation of mtTFA inhibits their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). {yields} Knockdown of mtTFA expression induces p21-dependent G1 cell cycle arrest. -- Abstract: Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

  13. Human epithelial growth factor receptor 2[Ile655Val] polymorphism and risk of breast fibroadenoma.

    Science.gov (United States)

    Zubor, Pavol; Kajo, Karol; Stanclova, Andrea; Szunyogh, Norbert; Galo, Silvester; Dussan, Carlos A; Minarik, Gabriel; Visnovsky, Jozef; Danko, Jan

    2008-02-01

    Studies on the association between the Ile to Val polymorphism at codon 655 of the human epithelial growth factor receptor 2 (HER-2) gene and susceptibility to breast cancer have been reported for almost all ethnic populations, with both positive or negative conclusions. No study, however, has yet been focused on the possible association between this gene and its predisposition to benign breast lesions, especially on risk for fibroadenoma. We aimed to study the association of the single nucleotide polymorphism V655 HER-2 gene polymorphism with histologically verified breast fibroadenoma risk. We conducted a molecular epidemiological case-control study of 70 breast fibroadenoma cases without cellular atypia and 172 healthy female controls. We found that the Val variant allele and genotype frequency of this polymorphism is higher in cases with fibroadenoma; however, this difference was not significant (allele Val 655: 27.86 and 22.67% in fibroadenoma and controls, respectively; genotype Ile/Val: 35.71 and 38.37% and Val/Val: 10.0 and 3.49% in fibroadenoma and controls, respectively). Applying logistic regression analysis, we found an increased risk of fibroadenoma formation in carriers of the Val allele (odds ratio = 1.17; 95% confidence interval = 0.67-2.05), in which the highest risk was associated with homozygous genotype (odds ratio = 3.07; 95% confidence interval = 0.97-9.72), but this risk was not significant. Stratification by age (cut-off 45 years) revealed the highest risk of fibroadenoma among young women homozygous for the Val allele (odds ratio = 3.30). The risk, however, was slightly increased (odds ratio = 1.24) among older carriers of the aberrant allele in their genotype as well, but it was not significant. In spite of insignificant differences, our results indicate that HER-2 Ile655Val polymorphism, especially in a homozygous form might play some role in the etiology of breast fibroadenoma formation. The significance of this susceptibility, however

  14. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  15. Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation

    OpenAIRE

    2012-01-01

    It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the...

  16. Human epidermal growth factor receptor-2 in oesophageal cancers: An observational study

    Institute of Scientific and Technical Information of China (English)

    Hazem Al-Momani; Rachel Barnes; Ahmed El-Hadi; Rachit Shah; Wyn G Lewis; Paul Edwards

    2012-01-01

    AIM:To determine the incidence of human epidermal growth factor receptor 2 (HER2) over expression in oesophageal cancers.METHODS:A retrospective study,of one hundred consecutive cases of endoscopic histological samples of oesophageal cancers from a single British cancer network were included.Cancer cases were diagnosed between April 2007 and June 2010.HER2 over expression was assessed using immunohistochemistry,those that scored "0" and "+1" were considered "negative"for HER2; those that scored "+3" were considered "Positive".Cases that were scored "+2" on immunohistochemistry further went on to have HER2 gene analysis using the Ventana HER brightfield dual-colour in situ hybridisations (HER B DISH) assay and either came back to be positive or negative for HER2 over expression.Overall survival was measured from date of histological diagnosis until date of death.93% of the cases were followed up till five years or death,and all were followed up till two years.Cases of gastro-oesophageal junctional tumours were excluded.RESULTS:The median age of our sample was 66years (range:38-91 years).Eighty one were male and 19 female.Ninety-one of the cases were adenocarcinoma of the oesophagus and the rest were cases of squamous cell carcinoma.The anatomical distribution of the tumours was; upper oesophagus 2,middle oesophagus 11,and 87 were in the lower oesophagus.Operative resection was completed in 15 cases; seven cases had attempted surgical resections,i.e.,open and close,33 patients received definitive chemo-radiation and 52 had palliative treatment.Twenty-five of the cancers showed evidence of HER2 over expression,all were adenocarcinomas.Of the 25 cases that showed evidence of HER2 over expression,21 (84%) were located in the lower third of the oesophagus.On staging,24 out of the 25 HER2 positive cases were at stage 3or more (13 at stage 3 and 11 at stage 4),For HER2 negative cases 37 were at stage 3 and 32 were staged as stage 4.Seventeen out of twenty five cases

  17. Human Genome-Wide RNAi Screen for Host Factors That Modulate Intracellular Salmonella Growth

    OpenAIRE

    Thornbrough, Joshua M.; Tom Hundley; Raphael Valdivia; Worley, Micah J.

    2012-01-01

    Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to ...

  18. The characteristics of human antibody targeting the Epidermal Growth Factor Receptor in vivo for radioimmunotherapy in a small animal model

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun Jung; Choi, Tae Hyun; Kim, Byoung Soo; Cheon, Gi Jeong [Korea Institue of Radiological and Medical Sciences, Seoul (Korea, Republic of); Hong, Kwang Won; Chang, Ki Hwan; Shin, Yong Won; Ryoo, Kyung Hwan; Shin, Yong Nam; Kim, Se Ho [Green Cross Corp., Yongin (Korea, Republic of)

    2010-05-15

    The identification of epidermal growth factor receptor (EGFR) as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including Erbitux for colon cancer. Many therapeutic approaches are aimed at the EGFR. Erbitux is example of monoclonal antibody inhibitors. The monoclonal antibodies block the extracellular ligand binding domain. EGFR4-2, IgG human monoclonal antibody, has been developed on the basis of human antibody gene library in Green Cross Corp. Small animal imaging is useful for preclinical evaluation of radiolabeled antibody to see biodistribution and targeting ability at serial time points in same animals

  19. Interleukin-8 increases vascular endothelial growth factor and neuropilin expression and stimulates ERK activation in human pancreatic cancer.

    Science.gov (United States)

    Li, Min; Zhang, Yuqing; Feurino, Louis W; Wang, Hao; Fisher, William E; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi

    2008-04-01

    Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor(165), and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy.

  20. Capillary growth in human skeletal muscle: physiological factors and the balance between pro-angiogenic and angiostatic factors

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Hoier, Birgitte

    2014-01-01

    In human skeletal muscle, the capillary net readily adapts according to the level of muscular activity to allow for optimal diffusion conditions for oxygen from the blood to the muscle. Animal studies have demonstrated that stimulation of capillary growth in skeletal muscle can occur either...... by mechanical or by chemical signalling. Mechanical signals originate from shear stress forces on the endothelial cell layer induced by the blood flowing through the vessel, but include also mechanical stretch and compression of the vascular structures and the surrounding tissue, as the muscle contracts...

  1. Elevated D-glucose concentrations modulate TGF-beta 1 synthesis by human cultured renal proximal tubular cells. The permissive role of platelet-derived growth factor.

    OpenAIRE

    Phillips, A.O.; Steadman, R.; Topley, N; Williams, J. D.

    1995-01-01

    Interstitial fibrosis is a marker of progression of renal impairment in diabetic nephropathy. Transforming growth factor (TGF)-beta 1 is one of a group of pro-fibrotic cytokines and growth factors that have been associated with the development of interstitial fibrosis. We have examined the modulating influence of glucose on the production of TGF-beta 1 by cultured human proximal tubular cells. Incubation of growth-arrested human proximal tubular cells (HPTC) (72 hours in serum free medium) in...

  2. Fibroblast growth factor receptors as therapeutic targets in human melanoma: synergism with BRAF inhibition.

    Science.gov (United States)

    Metzner, Thomas; Bedeir, Alexandra; Held, Gerlinde; Peter-Vörösmarty, Barbara; Ghassemi, Sara; Heinzle, Christine; Spiegl-Kreinecker, Sabine; Marian, Brigitte; Holzmann, Klaus; Grasl-Kraupp, Bettina; Pirker, Christine; Micksche, Michael; Berger, Walter; Heffeter, Petra; Grusch, Michael

    2011-10-01

    Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

  3. FGF growth factor analogs

    Science.gov (United States)

    Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY; Takahashi, Kazuyuki [Germantown, MD

    2012-07-24

    The present invention provides a fibroblast growth factor heparin-binding analog of the formula: ##STR00001## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, X, Y and Z are as defined, pharmaceutical compositions, coating compositions and medical devices including the fibroblast growth factor heparin-binding analog of the foregoing formula, and methods and uses thereof.

  4. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

    Science.gov (United States)

    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  5. Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

    Directory of Open Access Journals (Sweden)

    Rosa Adalberto L

    2011-07-01

    Full Text Available Abstract Background Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD, TGF-β1, and the combination of both factors (EMD+TGF-β1 on human osteoblastic cell cultures. Methods Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP, osteopontin (OPN and alkaline phosphatase (ALP immunodetection, total protein synthesis, ALP activity and bone-like nodule formation. Results All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group. Conclusions The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors in vitro supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.

  6. [Construction of recombinant human nerve growth factor (rh-β-NGF) eukaryotic vector and its expression in HEK293 cells].

    Science.gov (United States)

    Li, Jingchuan; Xue, Bofu; Yuan, Yuan; Ma, Mo; Zhu, Lin; Milburn, Rebecca; Le, Li; Hu, Peizhen; Ye, Jing

    2015-03-01

    Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.

  7. Trefoil factors are expressed in human and rat endocrine pancreas: differential regulation by growth hormone

    DEFF Research Database (Denmark)

    Jackerott, Malene; Lee, Ying C; Møllgård, Kjeld

    2006-01-01

    Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression o...... of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown....... of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic...... polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human...

  8. 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.

  9. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    Science.gov (United States)

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

  10. Expression of extracellular matrix components and related growth factors in human tendon and muscle after acute exercise

    DEFF Research Database (Denmark)

    Heinemeier, K M; Bjerrum, S S; Schjerling, P

    2013-01-01

    the patellar tendon and vastus lateralis muscle of each leg at 2 (n = 10), 6 (n = 11), or 26 h (n = 10) after exercise. Levels of messenger ribonucleic acid mRNA for collagens, noncollagenous matrix proteins, and growth factors were measured with real-time reverse transcription polymerase chain reaction......Acute kicking exercise induces collagen synthesis in both tendon and muscle in humans, but it is not known if this relates to increased collagen transcription and if other matrix genes are regulated. Young men performed 1 h of one-leg kicking at 67% of max workload. Biopsies were taken from....... In tendon, gene expression was unchanged except for a decrease in insulin-like growth factor-IEa (IGF-IEa; P ...

  11. PLACENTAL GROWTH FACTOR (PlGF) IS A POTENT VASODILATOR OF RAT AND HUMAN RESISTANCE ARTERIES

    Science.gov (United States)

    Osol, George; Celia, Gerard; Gokina, Natalia; Barron, Carolyn; Chien, Edward; Mandala, Maurizio; Luksha, Leonid; Kublickiene, Karolina

    2010-01-01

    The objectives of this study were to determine whether PlGF exerts a vasodilatory effect on rat uterine vessels (arcuate arteries and veins) and to examine regional differences in reactivity by comparing these responses to those of comparably-sized mesenteric vessels. We also sought to examine and compare its effects on human uterine and subcutaneous vessels. All vessels were studied in vitro, under pressurized (rat) or isometric wire-mounted (human) conditions, and exposed to a range of PlGF concentrations. Inhibitors of nitric oxide and prostaglandin synthesis were included in an effort to understand the causal mechanism(s). In rat uterine arteries, the effects of receptor inhibition and activation using selective ligands for VEGFR-1 (PlGF) vs. VEGFR-2 (VEGF-E) were determined, and real-time RT-PCR was performed to evaluate the effect of pregnancy on relative abundance of VEGFR-1 and VEGFR-2 message in the vascular wall. PlGF was a potent vasodilator of all vessels studied, with greatest sensitivity observed in rat uterine arteries. Pregnancy significantly augmented dilator sensitivity to PlGF, and this effect was associated with selective upregulation of VEGFR-1 message in the pregnant state. The contribution of nitric oxide was appreciable in rat and human uterine arteries, with lesser effects in rat uterine veins and mesenteric arteries, and with no observable effect in human subcutaneous vessels. Based on these results, we conclude that PlGF is a potent vasodilator of several vessel types in both humans and rats. Its potency and mechanism varies with physiological state and vessel location, and is mediated solely by the VEGFR-1 receptor subtype. Gestational changes in the uterine circulation suggest that this factor may play a role in modulating uterine vascular remodeling and blood flow during the pregnant state. PMID:18192215

  12. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

  13. New microbial growth factor

    Science.gov (United States)

    Bok, S. H.; Casida, L. E., Jr.

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a previously unknown microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight and has high specific activity. When added to the diets for a meadow-vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.

  14. Human conditions of insulin-like growth factor-I (IGF-I deficiency

    Directory of Open Access Journals (Sweden)

    Puche Juan E

    2012-11-01

    Full Text Available Abstract Insulin-like growth factor I (IGF-I is a polypeptide hormone produced mainly by the liver in response to the endocrine GH stimulus, but it is also secreted by multiple tissues for autocrine/paracrine purposes. IGF-I is partly responsible for systemic GH activities although it possesses a wide number of own properties (anabolic, antioxidant, anti-inflammatory and cytoprotective actions. IGF-I is a closely regulated hormone. Consequently, its logical therapeutical applications seems to be limited to restore physiological circulating levels in order to recover the clinical consequences of IGF-I deficiency, conditions where, despite continuous discrepancies, IGF-I treatment has never been related to oncogenesis. Currently the best characterized conditions of IGF-I deficiency are Laron Syndrome, in children; liver cirrhosis, in adults; aging including age-related-cardiovascular and neurological diseases; and more recently, intrauterine growth restriction. The aim of this review is to summarize the increasing list of roles of IGF-I, both in physiological and pathological conditions, underlying that its potential therapeutical options seem to be limited to those proven states of local or systemic IGF-I deficiency as a replacement treatment, rather than increasing its level upper the normal range.

  15. Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8.

    Science.gov (United States)

    Castellanos, Erick R; Ciferri, Claudio; Phung, Wilson; Sandoval, Wendy; Matsumoto, Marissa L

    2016-08-01

    Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.

  16. Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors.

    Science.gov (United States)

    Shalit, M; Sekhsaria, S; Malech, H L

    1995-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5

  17. Human monocyte-derived insulin-like growth factor-2 enhances the infection of human arterial endothelial cells by Chlamydia pneumoniae.

    Science.gov (United States)

    Lin, T M; Campbell, L A; Rosenfeld, M E; Kuo, C C

    2001-05-01

    It has been shown that infection of human endothelial cells by Chlamydia pneumoniae is enhanced by co-culturing endothelial cells with human monocytes and is mediated by monocyte-derived soluble factors. This study was conducted to identify the infectivity-enhancing factor. Serum-free conditioned medium of human monocytic cells was fractionated by ultrafiltration. The enhancing activity was found in the fraction in the molecular mass range between 5000 and 10,000 kDa. Recombinant human insulin-like growth factor (IGF)-1 or -2, with a molecular mass of 7500 kDa, was added to the culture medium of human endothelial cells for growing C. pneumoniae. Only IGF-2 enhanced C. pneumoniae growth. Pretreatment of the conditioned medium with a monoclonal antibody against IGF-2 blocked the enhancing activity. This suggests that the infectivity-enhancing factor is IGF-2 and that paracrine interactions between monocytes and endothelial cells in vivo can induce secretory products and sustain infection with C. pneumoniae within atherosclerotic lesions.

  18. Vascular endothelial growth factor receptor-1 in human cancer: concise review and rationale for development of IMC-18F1 (Human antibody targeting vascular endothelial growth factor receptor-1).

    Science.gov (United States)

    Schwartz, Jonathan D; Rowinsky, Eric K; Youssoufian, Hagop; Pytowski, Bronislaw; Wu, Yan

    2010-02-15

    The human vascular endothelial growth factor receptor-1 (VEGFR-1, or Flt-1) is widely expressed in normal and pathologic tissue and contributes to the pathogenesis of both neoplastic and inflammatory diseases. In human cancer, VEGFR-1 mediated signaling is responsible for both direct tumor activation and angiogenesis. VEGFR-1 mediated activation of nonmalignant supporting cells, particularly stromal, dendritic, hematopoietic cells, and macrophages, is also likely important for cancer pathogenesis. VEGFR-1 is also hypothesized to enable the development of cancer metastases by means of activation and premetastatic localization in distant organs of bone marrow-derived hematopoietic progenitor cells, which express VEGFR-1. IMC-18F1 is a fully human IgG(1) antibody that binds to VEGFR-1 and has been associated with the inhibition of cancer growth in multiple in vitro and human tumor xenograft models. The preliminary results of phase 1 investigations have also indicated a favorable safety profile for IMC-18F1 at doses that confer antibody concentrations that are associated with relevant antitumor activity in preclinical models.

  19. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  20. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

    2016-01-01

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  1. Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

    Energy Technology Data Exchange (ETDEWEB)

    Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

    2011-10-14

    Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

  2. Growth differentiation factor 8 suppresses cell proliferation by up-regulating CTGF expression in human granulosa cells.

    Science.gov (United States)

    Chang, Hsun-Ming; Pan, Hui-Hui; Cheng, Jung-Chien; Zhu, Yi-Min; Leung, Peter C K

    2016-02-15

    Connective tissue growth factor (CTGF) is a matricellular protein that plays a critical role in the development of ovarian follicles. Growth differentiation factor 8 (GDF8) is mainly, but not exclusively, expressed in the mammalian musculoskeletal system and is a potent negative regulator of skeletal muscle growth. The aim of this study was to investigate the effects of GDF8 and CTGF on the regulation of cell proliferation in human granulosa cells and to examine its underlying molecular determinants. Using dual inhibition approaches (inhibitors and small interfering RNAs), we have demonstrated that GDF8 induces the up-regulation of CTGF expression through the activin receptor-like kinase (ALK)4/5-mediated SMAD2/3-dependent signaling pathways. In addition, the increase in CTGF expression contributes to the GDF8-induced suppressive effect on granulosa cell proliferation. Our findings suggest that GDF8 and CTGF may play critical roles in the regulation of proliferative events in human granulosa cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    Science.gov (United States)

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

  4. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

    Science.gov (United States)

    Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  5. CCL5/CCR5 axis induces vascular endothelial growth factor-mediated tumor angiogenesis in human osteosarcoma microenvironment.

    Science.gov (United States)

    Wang, Shih-Wei; Liu, Shih-Chia; Sun, Hui-Lung; Huang, Te-Yang; Chan, Chia-Han; Yang, Chen-Yu; Yeh, Hung-I; Huang, Yuan-Li; Chou, Wen-Yi; Lin, Yu-Min; Tang, Chih-Hsin

    2015-01-01

    Chemokines modulate angiogenesis and metastasis that dictate cancer development in tumor microenvironment. Osteosarcoma is the most frequent bone tumor and is characterized by a high metastatic potential. Chemokine CCL5 (previously called RANTES) has been reported to facilitate tumor progression and metastasis. However, the crosstalk between chemokine CCL5 and vascular endothelial growth factor (VEGF) as well as tumor angiogenesis in human osteosarcoma microenvironment has not been well explored. In this study, we found that CCL5 increased VEGF expression and production in human osteosarcoma cells. The conditioned medium (CM) from CCL5-treated osteosarcoma cells significantly induced tube formation and migration of human endothelial progenitor cells. Pretreatment of cells with CCR5 antibody or transfection with CCR5 specific siRNA blocked CCL5-induced VEGF expression and angiogenesis. CCL5/CCR5 axis demonstrably activated protein kinase Cδ (PKCδ), c-Src and hypoxia-inducible factor-1 alpha (HIF-1α) signaling cascades to induce VEGF-dependent angiogenesis. Furthermore, knockdown of CCL5 suppressed VEGF expression and attenuated osteosarcoma CM-induced angiogenesis in vitro and in vivo. CCL5 knockdown dramatically abolished tumor growth and angiogenesis in the osteosarcoma xenograft animal model. Importantly, we demonstrated that the expression of CCL5 and VEGF were correlated with tumor stage according the immunohistochemistry analysis of human osteosarcoma tissues. Taken together, our findings provide evidence that CCL5/CCR5 axis promotes VEGF-dependent tumor angiogenesis in human osteosarcoma microenvironment through PKCδ/c-Src/HIF-1α signaling pathway. CCL5 may represent a potential therapeutic target against human osteosarcoma.

  6. [Influence of ASODN to the human tenon's fibroblasts in expressing CTGF induced by transforming growth factor beta2].

    Science.gov (United States)

    Hu, Yi-Zhen; Wang, Yu-Hong; Cao, Yang; Zhang, Ming-Chang

    2008-02-01

    To investigate the effect of connective tissue growth factor's antisense oligonucleotides (ASODN) on the growth of human tenon' s capsule fibroblasts (HTF) induced by transforming growth factor beta2 (TGF-beta2) in vitro. It was a experimental study. HTF was collected from glaucoma patients and cultured. The 5-6 passage was used for experiments. The HTF induced by TGF-beta2 was divided into the following groups: N group: normal HTF; T group: HTF induced by TGF-beta2; A group: CTGF ASODN antisense:5'-TACTGGCGGCGGTCAT-3' encapsulated with liposome; S group: sense 5'-ATGACCGCCGCCAGTA-3' encapsulated with liposome; D group: HTF encapsulated with liposome only. The activity of HTF treated by different concentrations of liposome was detected using methylthianolyldiphenyl tetrazolium bromide (MT) colorimetry. The expression of CTGF was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry assays. The expression of fibronectin (Fn) was examined by Western blot and immunocytochemistry assays. Liposome-ASODN (A group) significantly (F=15.25, 204.88, 19.73, 90.00; P HTF induced by TGF-beta2 compared with S and D group. However, Liposome alone (T group) has no significant impact in HTF growth compared with T group (t = 0.90, 2.32, 0.75, 2.11; P > 0.05). CTGF-ASODN inhibits the CTGF and Fn expression of HTF induced by TGF-beta2, which may delay the formation of scar in glaucoma filtering surgery.

  7. Initial Biochemical Characterization of Cells Derived from Human Periodontium and Their In vitro Response to Platelet-Derived Growth Factor, Epidermal Growth Factor and Transforming Growth Factor-Beta

    Science.gov (United States)

    1988-05-01

    lipolysis (Van Wyk, 1984). From a systemic standpoint, the in vivo effects of insulin-like growth factors can essentially be broken into insulin-like...of lean and obese mice: comparison with insulin. Endocrinology. 105:72j-730. Polson, A. and Zander, H. 1974. J. Periodontol. 45:726 Polson, A.M. and

  8. Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines.

    Science.gov (United States)

    Blanchère, M; Saunier, E; Mestayer, C; Broshuis, M; Mowszowicz, I

    2002-11-01

    TGF beta can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGF beta function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGF beta 1 and TGF beta 2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGF beta 1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGF beta secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGF beta which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGF beta secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGF beta, and to regulate this secretion through stromal-epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.

  9. Insulin-like growth factors and the multiplication of Tera-2, a human teratoma-derived cell line.

    Science.gov (United States)

    Biddle, C; Li, C H; Schofield, P N; Tate, V E; Hopkins, B; Engstrom, W; Huskisson, N S; Graham, C F

    1988-07-01

    A human teratoma cell line (Tera-2) was grown in serum-free medium, and the population multiplication was stimulated by the addition of somatomedins/insulin-like growth factors (IGFs). Both IGF-I and IGF-II gave maximal stimulation when added daily at 10 ng ml-1. The IGFs did not substantially change the labelling index of the cells, and the IGFs appeared to exert their effect on population multiplication by increasing cell survival. Membranes isolated from Tera-2 cells displayed both type 1 and type 2 IGF receptors.

  10. Post-transcriptional regulation of neurofibromin level in cultured human melanocytes in response to growth factors.

    Science.gov (United States)

    Griesser, J; Kaufmann, D; Maier, B; Mailhammer, R; Kuehl, P; Krone, W

    1997-03-01

    Among the symptoms that characterize neurofibromatosis type 1 (NF1) are pigmentation anomalies such as cafe au lait spots. It has been suggested that the reduction of the neurofibromin level in the epidermis of NF1 patients is responsible for the observed signs such as altered melanogenesis and altered density of melanocytes. Our studies show that in cultured normal human melanocytes, the neurofibromin level can be varied in vitro over a wide range by using different culture conditions. The influence of factors that control differentiation and proliferation of melanocytes on neurofibromin levels was studied. Immunoprecipitation followed by western blotting showed a 3- to 4-fold increase of neurofibromin after stimulation by PMA or bFGF, respectively, and a 1.5-fold increase in cells stimulated with steel factor. The increase of neurofibromin was not paralleled by a higher NF1 mRNA level as proved by northern blotting. Pulse-chase experiments with 35S-labeled melanocytes revealed an approximately 3-fold increase in the half-life of neurofibromin in bFGF- or PMA-stimulated cells compared to controls. These results indicate that the neurofibromin level of cultured melanocytes can be regulated by a mechanism independent of NF1 gene transcription and translation, which might influence the degradation rate of the protein.

  11. [Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells].

    Science.gov (United States)

    Yin, Kai; Ma, Li; Shen, Chuan'an; Shang, Yuru; Li, Dawei; Li, Longzhu; Zhao, Dongxu; Cheng, Wenfeng

    2016-05-01

    To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture

  12. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in hindlimb ischemia of dogs

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A preclinical study of treating peripheral artery occlusive disease (PAD) was performed by using a hepatocyte growth factor (HGF) gene-expressing vector, plasmid pUDKH, in a dog model with complete ischemia of one hindlimb. After ligation of femoral artery of one hindlimb, pUDKH was transferred directly into the ischemic limb muscles. The angiogenic activity of the plasmid pUDKH was evaluated. On D 30 after injecting once of pUDKH at different doses into local muscles immediately after operation, the degree of augmentation of collateral vessel formation was significantly greater than that treated by blank vector. In addition, the blood flow rate of femoral artery in dogs treated with pUDKH was recovered on D 90, while the flow rate was only 1/5 to 1/3 in control dogs. The pulse amplitude of pUDKH-treated dogs was recovered on D 90, but it was hardly detectable in most of the control dogs. The side effects of intramuscular transfection of pUDKH were also investigated, and no significant positive change was found. It is suggested that angiogenesis induced by HGF gene has the potential for clinical use in the treatment of peripheral arterial diseases.

  13. Expression of a Functional Recombinant Human Basic Fibroblast Growth Factor from Transgenic Rice Seeds

    Directory of Open Access Journals (Sweden)

    Daichang Yang

    2013-02-01

    Full Text Available Basic fibroblast growth factor (FGF-2 is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF. An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins.

  14. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    Directory of Open Access Journals (Sweden)

    Lucília Pereira da Silva

    2014-01-01

    Full Text Available Fibroblasts colonization into injured areas during wound healing (WH is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH.

  15. 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

    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...... 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...

  16. Interferon alpha2 recombinant and epidermal growth factor modulate proliferation and hypusine synthesis in human epidermoid cancer KB cells.

    Science.gov (United States)

    Caraglia, M; Passeggio, A; Beninati, S; Leardi, A; Nicolini, L; Improta, S; Pinto, A; Bianco, A R; Tagliaferri, P; Abbruzzese, A

    1997-06-15

    We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

  17. The effect of Isosorbide Dinitrate on vascular endothelial growth factor production by human leukemic cell lines in vitro

    Directory of Open Access Journals (Sweden)

    Hajighasemi F

    2009-03-01

    Full Text Available "nBackground: Vascular endothelial growth factor (VEGF has mitogenic effect for endothelial cells and is an important mediator of tumor expansion, metastasis and angiogenesis in vivo. Isosorbide dinitrate, as a nitric oxide donor, has been widely used in treatment of many cardiovascular diseases such as congestive heart failure and acute coronary syndromes. Furthermore this drug was found to have inhibitory effect on angiogenesis, tumor growth and metastasis in vivo. In the present study we evaluated the isosorbide effect on the VEGF production using some human leukemic cell lines. "nMethods: Human leukemic MOLT-4, JURKAT and U937 cells were cultured in complete RPMI medium. The cells at the exponential growth phase were then incubated with different concentrations of Isosorbide (4´10-7 -4´10-4 M in the presence or absence of PMA (25ng/ml for 24 hours. The VEGF concentrations in the culture supernatants were measured by enzyme immunoassay kits (R&D systems according to the manufacturer's instructions. "nResults: The level of VEGF produced by the human leukemic cell lines which was treated with different concentrations of isosorbide, did not show any significant difference with untreated control cells. "nConclusions: The results of this study showed that isosorbide had no significant effect on VEGF production. Our findings suggest that anti-angiogenesis effect of isosorbide could be mediated through VEGF-independent mechanism(s. Further studies are warranted to determine definite isosorbide effect on VEGF and other angiogenic factors production in patients as well as animal models.

  18. Microvesicles derived from human Wharton's jelly mesenchymal stem cells promote human renal cancer cell growth and aggressiveness through induction of hepatocyte growth factor.

    Directory of Open Access Journals (Sweden)

    Tao Du

    Full Text Available In our previous study, microvesicles (MVs released from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC. By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0 was assessed. Cell counting kit-8 (CCK-8 assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro or examination of cyclin D1 expression (in vivo was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro or MMP-2 and MMP-9 expression (in vivo. AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.

  19. Effect of recombinant human platelet-derived growth factor-BB-coated sutures on Achilles tendon healing in a rat model: A histological and biomechanical study

    Directory of Open Access Journals (Sweden)

    Stephen H Cummings

    2012-12-01

    Full Text Available Purpose: Repairing tendon injuries with recombinant human platelet-derived growth factor-BB has potential for improving surgical outcomes. Augmentation of sutures, a critical component of surgical tendon repair, by coating with growth factors may provide a clinically useful therapeutic device for improving tendon repair. Therefore, the purpose of this study was to (a coat Vicryl sutures with a defined dose of recombinant human platelet-derived growth factor-BB without additional coating excipients (e.g. gelatin, (b quantify the recombinant human platelet-derived growth factor-BB released from the suture, and (c use the recombinant human platelet-derived growth factor-BB-coated sutures to enhance tendon repair in a rat Achilles tendon transection model. Methods: Vicryl sutures were coated with 0, 0.3, 1.0, and 10.0 mg/mL concentrations of recombinant human platelet-derived growth factor-BB using a dip-coating process. In vitro release was quantified by an enzyme-linked immunosorbent assay. Acutely transected rat Achilles tendons were repaired using one of the four suture groups (n = 12 per group. Four weeks following repair, the tensile biomechanical and histological (i.e. collagen organization and angiogenesis properties were determined. Results: A dose-dependent bolus release of recombinant human platelet-derived growth factor-BB occurred within the first hour in vitro, followed by a gradual release over 48 h. There was a significant increase in ultimate tensile strength (p < 0.01 in the two highest recombinant human platelet-derived growth factor-BB dose groups (1.9 ± 0.5 and 2.1 ± 0.5 MPa relative to controls (1.0 ± 0.2 MPa. The modulus significantly increased (p = 0.031 with the highest recombinant human platelet-derived growth factor-BB dose group (7.2 ± 3.8 MPa relative to all other groups (control: 3.5 ± 0.9 MPa. No significant differences were identified for the maximum load or stiffness. The histological collagen and angiogenesis

  20. /sup 125/I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of /sup 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 AB diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more /sup 125/I-hEGF than did fetal membranes (P<0.0001). There was no significant differnce in /sup 125/I-hEGF binding to fetal membranes from the various pregnancy states (P<0.05). /sup 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<0.05). The binding to placentas from pregnancies complicated by White class AB diabetes or large for gestational age infants, on the other hand, was not significantly different from that to placentas from normal and appropriate for gestational age pregnancies. /sup 125/I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P<0.05). Placental and fetal membrane /sup 125/I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P<0.05). Placental but not fetal membrane /sup 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.

  1. [Morphological study on development of nerve growth factor-positive neurons in the cerebellum of human fetus].

    Science.gov (United States)

    Zheng, Lan-Rong; Shao, Jin-Gui

    2012-02-01

    To investigate the growth and development of nerve growth factor (NGF)-positive neurons in the cerebellum of midanaphase human fetus. The expression of the NGF-positive neurons in the cerebrum of human fetus was observed by immunohistochemical methods, and the integral absorbance (IA) was detected. By the 3rd to 4th month of gestation, neurons was seen in the ependymal, central, and marginal plate of cerebellum; the nucleus was oval and the neurons had short and small processes. By the 5th to 7th month of gestation, the number of NGF-positive neurons increased, the expressions enhanced, the nucleus was round-, oval-, or fusiform-shaped, the neurons grew larger in size, and the Purkinje cells showed NGF-positive expression. By the 8th to 10th month of gestation, the NGF-positive expression was enhanced with deeper dying, the body of Purkinje cells grew larger gradually, and the number of NGF-positive neurons in the granular cell layer and molecular layer increased. IA of the cerebellar cortical neurons of the 3rd, 4th, 5th, 6th, 7th, and 8th month of gestation showed an increasing trend, and significant difference was observed (P positive neurons in the cerebellum play an important role for differentiation, proliferation, migration, and growth of neurons in the cerebellum.

  2. Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Guo Yingqiang

    2010-05-01

    Full Text Available Abstract Background Atherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF is a member of the vascular endothelial growth factor (VEGF family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs and smooth muscle cells (VSMCs. Results In growth-arrested human VECs and VSMCs, Ang II induced PlGF mRNA expression after 4 hour treatment, and peaked at 24 hours. 10-6 mol/L Ang II increased PlGF protein production after 8 hour treatment, and peaked at 24 hours. Stimulation with Ang II also induced mRNA expression of VEGF receptor-1 and -2(VEGFR-1 and -2 in these cells. The Ang II type I receptor (AT1R antagonist blocked Ang II-induced PlGF gene expression and protein production. Several intracellular signals elicited by Ang II were involved in PlGF synthesis, including activation of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2 and PI3-kinase. A neutralizing antibody against PlGF partially inhibited the Ang II-induced proliferation of VECs and VSMCs. However, this antibody showed little effect on the basal proliferation in these cells, whereas blocking antibody of VEGF could suppress both basal and Ang II-induced proliferation in VECs and VSMCs. Conclusion Our results showed for the first time that Ang II could induce the gene expression and protein production of PlGF in VECs and VSMCs, which might play an important role in the pathogenesis of vascular inflammation and atherosclerosis.

  3. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  4. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  5. Local administration of insulin-like growth factor-I (IGF-I) stimulates tendon collagen synthesis in humans

    DEFF Research Database (Denmark)

    Hansen, Mette; Boesen, A; Holm, L

    2012-01-01

    Collagen is the predominant structural protein in tendons and ligaments, and can be controlled by hormonal changes. In animals, injections of insulin-like growth factor I (IGF-I) has been shown to increase collagen synthesis in tendons and ligaments and to improve structural tissue healing......, but the effect of local IGF-I administration on tendon collagen synthesis in human has not been studied. The purpose of this study was to study whether local injections of IGF-I would have a stimulating effect on tendon collagen synthesis. Twelve healthy nonsmoking men [age 62 ± 1 years (mean ± SEM), BMI 27 ± 1......] participated. Two injections of either human recombinant IGF-I (0.1 mL Increlex©) or saline (control) into each patellar tendon were performed 24-h apart, respectively. Tendon collagen fractional synthesis rate (FSR) was measured by stable isotope technique in the hours after the second injection...

  6. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  7. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Zhu, Qingchun; Dong, Rui; Zhao, Chengcheng; Li, Guoling; Li, Guo; Gao, Wenchao; Jiang, Gelong; Zheng, Enqin; Cai, Gengyuan; Moisyadi, Stefan; Urschitz, Johann; Yang, Huaqiang; Liu, Dewu; Wu, Zhenfang

    2017-01-01

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins. PMID:28117418

  8. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors.

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Zhu, Qingchun; Dong, Rui; Zhao, Chengcheng; Li, Guoling; Li, Guo; Gao, Wenchao; Jiang, Gelong; Zheng, Enqin; Cai, Gengyuan; Moisyadi, Stefan; Urschitz, Johann; Yang, Huaqiang; Liu, Dewu; Wu, Zhenfang

    2017-01-24

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

  9. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  10. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zehai; LIU Xinyue; ZOU Ping

    2007-01-01

    This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concen- trations (12.5-200 μmol/L) for different time lengths (12-48 h). The proliferation of the U937 leu- kemic cells was determined by MTT assay. Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resvera-trol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

  11. Genetic factors associated with small for gestational age birth and the use of human growth hormone in treating the disorder

    Directory of Open Access Journals (Sweden)

    Saenger Paul

    2012-05-01

    Full Text Available Abstract The term small for gestational age (SGA refers to infants whose birth weights and/or lengths are at least two standard deviation (SD units less than the mean for gestational age. This condition affects approximately 3%–10% of newborns. Causes for SGA birth include environmental factors, placental factors such as abnormal uteroplacental blood flow, and inherited genetic mutations. In the past two decades, an enhanced understanding of genetics has identified several potential causes for SGA. These include mutations that affect the growth hormone (GH/insulin-like growth factor (IGF-1 axis, including mutations in the IGF-1 gene and acid-labile subunit (ALS deficiency. In addition, select polymorphisms observed in patients with SGA include those involved in genes associated with obesity, type 2 diabetes, hypertension, ischemic heart disease and deletion of exon 3 growth hormone receptor (d3-GHR polymorphism. Uniparental disomy (UPD and imprinting effects may also underlie some of the phenotypes observed in SGA individuals. The variety of genetic mutations associated with SGA births helps explain the diversity of phenotype characteristics, such as impaired motor or mental development, present in individuals with this disorder. Predicting the effectiveness of recombinant human GH (hGH therapy for each type of mutation remains challenging. Factors affecting response to hGH therapy include the dose and method of hGH administration as well as the age of initiation of hGH therapy. This article reviews the results of these studies and summarizes the success of hGH therapy in treating this difficult and genetically heterogenous disorder.

  12. Genetic factors associated with small for gestational age birth and the use of human growth hormone in treating the disorder.

    Science.gov (United States)

    Saenger, Paul; Reiter, Edward

    2012-05-15

    The term small for gestational age (SGA) refers to infants whose birth weights and/or lengths are at least two standard deviation (SD) units less than the mean for gestational age. This condition affects approximately 3%-10% of newborns. Causes for SGA birth include environmental factors, placental factors such as abnormal uteroplacental blood flow, and inherited genetic mutations. In the past two decades, an enhanced understanding of genetics has identified several potential causes for SGA. These include mutations that affect the growth hormone (GH)/insulin-like growth factor (IGF)-1 axis, including mutations in the IGF-1 gene and acid-labile subunit (ALS) deficiency. In addition, select polymorphisms observed in patients with SGA include those involved in genes associated with obesity, type 2 diabetes, hypertension, ischemic heart disease and deletion of exon 3 growth hormone receptor (d3-GHR) polymorphism. Uniparental disomy (UPD) and imprinting effects may also underlie some of the phenotypes observed in SGA individuals. The variety of genetic mutations associated with SGA births helps explain the diversity of phenotype characteristics, such as impaired motor or mental development, present in individuals with this disorder. Predicting the effectiveness of recombinant human GH (hGH) therapy for each type of mutation remains challenging. Factors affecting response to hGH therapy include the dose and method of hGH administration as well as the age of initiation of hGH therapy. This article reviews the results of these studies and summarizes the success of hGH therapy in treating this difficult and genetically heterogenous disorder.

  13. Human tumor cells induce angiogenesis through positive feedback between CD147 and insulin-like growth factor-I.

    Directory of Open Access Journals (Sweden)

    Yanke Chen

    Full Text Available Tumor angiogenesis is a complex process based upon a sequence of interactions between tumor cells and endothelial cells. Previous studies have shown that CD147 was correlated with tumor angiogenesis through increasing tumor cell secretion of vascular endothelial growth factor (VEGF and matrix metalloproteinases (MMPs. In this study, we made a three-dimensional (3D tumor angiogenesis model using a co-culture system of human hepatocellular carcinoma cells SMMC-7721 and humanumbilical vein endothelial cells (HUVECs in vitro. We found that CD147-expressing cancer cells could promote HUVECs to form net-like structures resembling the neo-vasculature, whereas the ability of proliferation, migration and tube formation of HUVECs was significantly decreased in tumor conditioned medium (TCM of SMMC-7721 cells transfected with specific CD147-siRNA. Furthermore, by assaying the change of pro-angiogenic factors in TCM, we found that the inhibition of CD147 expression led to significant decrease of VEGF and insulin-like growth factor-I (IGF-I secretion. Interestingly, we also found that IGF-I up-regulated the expression of CD147 in both tumor cells and HUVECs. These findings suggest that there is a positive feedback between CD147 and IGF-I at the tumor-endothelial interface and CD147 initiates the formation of an angiogenesis niche.

  14. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Reichenbach, Andreas; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1

  15. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Moritz Veltmann

    Full Text Available Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE cells.Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5 expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 si

  16. Longitudinal Study of Cytokine Expression, Lipid Profile and Neuronal Growth Factors in Human Breast Milk from Term and Preterm Deliveries.

    Science.gov (United States)

    Collado, Maria Carmen; Santaella, Marina; Mira-Pascual, Laia; Martínez-Arias, Elena; Khodayar-Pardo, Parisá; Ros, Gaspar; Martínez-Costa, Cecilia

    2015-10-19

    Breast milk (BM) is considered as a reference for infant nutrition. The role of bioactive components, such as cytokines, hormones, growth factors (GFs) and fatty acids (FAs) is poorly known, but they might be implicated in immune response development. The aim of this study was to identify the lipid profile and the spectrum of cytokines and neuronal GF in BM samples and analyse the influence of gestational age and lactation time on these components. This study used a longitudinal prospective method for the characterization of cytokines, FAs and GFs global profiles in 120 BM samples from 40 healthy mothers (20 preterm and 20 term) collected as colostrum, transitional and mature milk. The cytokines were analysed by protein array (Ray Bio® Human Cytokine Array G6. Ray Biotech, Inc. Norcross, GA, USA) and the FAs were analysed by gas chromatography. The FA profile was similar between the term and the preterm BM samples. Omega-3-α-linoleic and docosahexaenoic acid (DHA) and omega-6-linoleic acid were the most abundant in the term and preterm samples during lactation. Omega-3 ETA and omega-3 EPA we observed exclusively in the preterm samples. The cytokine profile showed a different trend based on gestational age. A significantly higher expression of neurotrophic factors was found in the mature preterm milk samples as compared to the mature term samples. Our study is the first to identify the influence and interactions of perinatal factors on cytokine, GFs and FAs in human milk.

  17. Ex Vivo Gene Therapy Using Human Mesenchymal Stem Cells to Deliver Growth Factors in the Skeletal Muscle of a Familial ALS Rat Model.

    Science.gov (United States)

    Suzuki, Masatoshi; Svendsen, Clive N

    2016-01-01

    Therapeutic protein and molecule delivery to target sites by transplanted human stem cells holds great promise for ex vivo gene therapy. Our group has demonstrated the therapeutic benefits of ex vivo gene therapy targeting the skeletal muscles in a transgenic rat model of familial amyotrophic lateral sclerosis (ALS). We used human mesenchymal stem cells (hMSCs) and genetically modified them to release neuroprotective growth factors such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). Intramuscular growth factor delivery via hMSCs can enhance neuromuscular innervation and motor neuron survival in a rat model of ALS (SOD1(G93A) transgenic rats). Here, we describe the protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of hMSCs and hMSC transplantation into the skeletal muscle of a familial ALS rat model.

  18. Synthesis of insulin-like growth factor binding protein 3 in vitro in human articular cartilage cultures.

    Science.gov (United States)

    Eviatar, Tamar; Kauffman, Hannah; Maroudas, Alice

    2003-02-01

    To quantify the rate of synthesis of insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor 1 (IGF-1) by in vitro cultures of normal and osteoarthritic (OA) human articular cartilage. Levels of IGF-1 and IGFBP-3 in media from in vitro cultures of human cartilage were determined by radioimmunoassay (RIA). IGFBPs were characterized by immunoblots and ligand blots. Ultrafiltration and RIA analysis of synovial fluid (SF) samples and washings of cartilage samples ex vivo were used to calculate partition coefficients and to estimate the amount of IGF-1 and IGFBP-3 in cartilage in vivo. OA cartilage synthesized 150 ng of IGFBP-3 per gm of cartilage per day, compared with 50 ng synthesized by normal cartilage. The surface zone of normal cartilage produced more IGFBP-3 than did the deep zone. Immunoblots and ligand blots confirmed the presence of IGFBP-3. IGFBP-3 synthesis was stimulated by exogenous IGF-1. No freshly synthesized IGF-1 was detected. The quantities of IGF-1 and IGFBP-3 present ex vivo were 11.3 and 78.7 ng/gm of cartilage in normal cartilage and 21.6 and 225.4 ng/gm in OA cartilage. The results show that while IGFBP-3 is synthesized in explant cultures, IGF-1 is not. The rate of IGFBP-3 synthesis is 3 times higher in OA than in normal cartilage. Both IGFBP-3 and IGF-1 penetrate into cartilage from SF in vivo. We estimate that the quantities of IGFBP-3 produced in culture by human cartilage are small compared with the amount supplied in the form of "small complexes" from the circulation. The high value of the partition coefficient of IGFBP-3 implies binding to the matrix.

  19. Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

    Science.gov (United States)

    Dunand, C; Hoffmann, P; Sapin, V; Blanchon, L; Salomon, A; Sergent, F; Benharouga, M; Sabra, S; Guibourdenche, J; Lye, S J; Feige, J J; Alfaidy, N

    2014-09-01

    EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors. © 2014 by the Society for the Study of Reproduction, Inc.

  20. Role of iron in inactivation of epidermal growth factor receptor after asbestos treatment of human lung and pleural target cells.

    Science.gov (United States)

    Baldys, Aleksander; Aust, Ann E

    2005-05-01

    Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers. Here, we examined the effects of asbestos on the epidermal growth factor receptor (EGFR) in human lung epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial (SAEC) cells. Treatment of A549, MET5A, and SAEC cells with asbestos caused a significant reduction of EGFR tyrosine phosphorylation. This was both time- (15 min to 24 h) and concentration-dependent (1.5, 3, and 6 mug/cm(2)) in A549 cells. Also, treatment with 6 mug/cm(2) crocidolite for 24 h diminished the phosphorylation levels of human EGFR 2 (HER2). Exposure of A549 cells to 6 mug/cm(2) crocidolite for 3-24 h resulted in no detectable Y1045 phosphorylation and no apparent degradation of the EGFR. Inhibition of fiber endocytosis resulted in a considerable inhibition of EGFR dephosphorylation. Removal of iron from asbestos by desferrioxamine B or phytic acid inhibited asbestos-induced decreases in EGFR phosphorylation. The effects of crocidolite, amosite, and chrysotile on the EGFR phosphorylation state appeared to be directly related to the amount of iron mobilized from these fibers. These results strongly suggest that iron plays an important role in asbestos-induced inactivation of EGFR.

  1. Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus.

    Science.gov (United States)

    Hamady, Zaed Z R; Scott, Nigel; Farrar, Mark D; Lodge, J Peter A; Holland, Keith T; Whitehead, Terence; Carding, Simon R

    2010-04-01

    Human growth factors are potential therapeutic agents for various inflammatory disorders affecting the gastrointestinal tract. However, they are unstable when administered orally and systemic administration requires high doses increasing the risk of unwanted side effects. Live microorganism-based delivery systems can overcome these problems although they suffer from the inability to control heterologous protein production and there are concerns regarding biosafety and environmental contamination. To overcome these limitations we have developed a new live bacteria drug-delivery system using the human commensal gut bacterium Bacteroides ovatus engineered to secrete human growth factors in response to dietary xylan. The anaerobic nature of B ovatus provides an inherent biosafety feature. B ovatus strains expressing human keratinocyte growth factor-2, which plays a central role in intestinal epithelial homeostasis and repair (BO-KGF), were generated by homologous recombination and evaluated using the dextran sodium sulfate (DSS)-induced model of intestinal epithelial injury and colitis. In response to xylan BO-KGF produced biologically active KGF both in vitro and in vivo. In DSS treated mice administration of xylan and BO-KGF had a significant therapeutic effect in reducing weight loss, improving stool consistency, reducing rectal bleeding, accelerating healing of damaged epithelium, reducing inflammation and neutrophil infiltration, reducing expression of pro-inflammatory cytokines, and accelerating production of goblet cells. BO-KGF and xylan treatment also had a marked prophylactic effect limiting the development of inflammation and disruption of the epithelial barrier. This novel, diet-regulated, live bacterial drug delivery system may be applicable to treating various bowel disorders.

  2. High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.

    Science.gov (United States)

    Khalili, Mostafa; Soleyman, Mohammad Reza; Baazm, Maryam; Beyer, Cordian

    2015-07-01

    Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 μM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.

  3. Insulin-Like Growth Factor Binding Protein-6 Alters Skeletal Muscle Differentiation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Doaa Aboalola

    2017-01-01

    Full Text Available Insulin-like growth factor binding protein-6 (IGFBP-6, the main regulator of insulin-like growth factor-2 (IGF-2, is a component of the stem cell niche in developing muscle cells. However, its role in muscle development has not been clearly defined. In this study, we investigated the role of IGFBP-6 in muscle commitment and differentiation of human mesenchymal stem cells derived from the placenta. We showed that placental mesenchymal stem cells (PMSCs have the ability to differentiate into muscle cells when exposed to a specific culture medium by expressing muscle markers Pax3/7, MyoD, myogenin, and myosin heavy chain in a stage-dependent manner with the ultimate formation of multinucleated fibers and losing pluripotency-associated markers, OCT4 and SOX2. The addition of IGFBP-6 significantly increased pluripotency-associated markers as well as muscle differentiation markers at earlier time points, but the latter decreased with time. On the other hand, silencing IGFBP-6 decreased both pluripotent and differentiation markers at early time points. The levels of these markers increased as IGFBP-6 levels were restored. These findings indicate that IGFBP-6 influences MSC pluripotency and myogenic differentiation, with more prominent effects observed at the beginning of the differentiation process before muscle commitment.

  4. Calorie restriction decreases murine and human pancreatic tumor cell growth, nuclear factor-κB activation, and inflammation-related gene expression in an insulin-like growth factor-1-dependent manner.

    Directory of Open Access Journals (Sweden)

    Alison E Harvey

    Full Text Available Calorie restriction (CR prevents obesity and has potent anticancer effects that may be mediated through its ability to reduce serum growth and inflammatory factors, particularly insulin-like growth factor (IGF-1 and protumorigenic cytokines. IGF-1 is a nutrient-responsive growth factor that activates the inflammatory regulator nuclear factor (NF-κB, which is linked to many types of cancers, including pancreatic cancer. We hypothesized that CR would inhibit pancreatic tumor growth through modulation of IGF-1-stimulated NF-κB activation and protumorigenic gene expression. To test this, 30 male C57BL/6 mice were randomized to either a control diet consumed ad libitum or a 30% CR diet administered in daily aliquots for 21 weeks, then were subcutaneously injected with syngeneic mouse pancreatic cancer cells (Panc02 and tumor growth was monitored for 5 weeks. Relative to controls, CR mice weighed less and had decreased serum IGF-1 levels and smaller tumors. Also, CR tumors demonstrated a 70% decrease in the expression of genes encoding the pro-inflammatory factors S100a9 and F4/80, and a 56% decrease in the macrophage chemoattractant, Ccl2. Similar CR effects on tumor growth and NF-κB-related gene expression were observed in a separate study of transplanted MiaPaCa-2 human pancreatic tumor cell growth in nude mice. In vitro analyses in Panc02 cells showed that IGF-1 treatment promoted NF-κB nuclear localization, increased DNA-binding of p65 and transcriptional activation, and increased expression of NF-κB downstream genes. Finally, the IGF-1-induced increase in expression of genes downstream of NF-κB (Ccdn1, Vegf, Birc5, and Ptgs2 was decreased significantly in the context of silenced p65. These findings suggest that the inhibitory effects of CR on Panc02 pancreatic tumor growth are associated with reduced IGF-1-dependent NF-κB activation.

  5. Mechanisms in decorin regulation of vascular endothelial growth factor-induced human trophoblast migration and acquisition of endothelial phenotype.

    Science.gov (United States)

    Lala, Neena; Girish, Gannareddy V; Cloutier-Bosworth, Alia; Lala, Peeyush K

    2012-09-01

    Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.

  6. Muscle force and endurance in untreated and human growth hormone or insulin-like growth factor-I-treated patients with growth hormone deficiency or Laron syndrome.

    Science.gov (United States)

    Brat, O; Ziv, I; Klinger, B; Avraham, M; Laron, Z

    1997-01-01

    Muscle force and endurance of four muscle groups (biceps, triceps, hamstrings and quadriceps) were measured by a computerized device in three groups: (A) 4 boys with isolated growth hormone deficiencies (IGHD) examined before at 10 and 24 months of hGH treatment; (B) 5 children (2 F, 3 M) with Laron syndrome were examined 3.5-4 years after initiation of insulin-like growth factor-I (IGF-I) treatment, and (C) comprised 8 untreated adults (5 F, 3 M) with Laron syndrome. For each patient, 2 matched controls, by age, sex, physical activity and height below the 50th percentile, were examined. GH- or IGF-I-deficient patients before treatment revealed reduced muscle force and endurance. GH treatment (0.6 U/kg/week) restored muscle force and endurance, progressively, mainly in the boys with puberty. Three to 4 years of IGF-I treatment (150 micrograms/kg/day) in patients with Laron syndrome proved to have a weaker effect than GH in restoring muscle force. The difference in effectiveness between hGH and IGF-I in restoring muscle force may be due to either the more marked muscle underdevelopment in Laron syndrome patients than in patients with IGHD or a difference in action potential between the two hormones.

  7. Molecular docking and dynamic studies of human growth factor receptorbound protein (Grb 2 insights to identify novel inhibitors

    Directory of Open Access Journals (Sweden)

    Sandeep S

    2016-10-01

    Full Text Available Background: Human growth factor receptor bound protein-2 (Grb 2 involves in initiation of kinase signaling by Son of Sevenless (SOS and activates mitogen activated protein kinase pathway. Grb2 overexpress during cancerous condition hence it emerged as a potent target for various cancers. Material and Methods: Seven pharmacophores were developed from seven co-crystal structures of Grb2 and applied for common pharmacophore hypothesis. Two common pharmacophore hypothesis (CPH models were screened and hits were applied for docking and free energy [G] calculations. Results: Two leads were proposed from docking and G analysis. Energy of the system, RMSD, RMSF, hydrogen bonds and water bridges of lead1 was better than the co-crystal ligand during 50 ns molecular dynamics simulations. Discussion: Two leads are interacting with Src homology 2 (SH2 domain of Grb2 and blocking the function of Grb2.

  8. Hype, harmony and human factors: applying user-centered design to achieve sustainable telehealth program adoption and growth.

    Science.gov (United States)

    Rossos, P G; St-Cyr, O; Purdy, B; Toenjes, C; Masino, C; Chmelnitsky, D

    2015-01-01

    Despite decades of international experience with the use of information and communication technologies in healthcare delivery, widespread telehealth adoption remains limited and progress slow. Escalating health system challenges related to access, cost and quality currently coincide with rapid advancement of affordable and reliable internet based communication technologies creating unprecedented opportunities and incentives for telehealth. In this paper, we will describe how Human Factors Engineering (HFE) and user-centric elements have been incorporated into the establishment of telehealth within a large academic medical center to increase acceptance and sustainability. Through examples and lessons learned we wish to increase awareness of HFE and its importance in the successful implementation, innovation and growth of telehealth programs.

  9. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  10. Current status of anti-human epidermal growth factor receptor 2 therapies: predicting and overcoming herceptin resistance.

    Science.gov (United States)

    Chung, Alice; Cui, Xiaojiang; Audeh, William; Giuliano, Armando

    2013-08-01

    Human epidermal growth factor receptor 2-overexpressing (HER2+) breast cancer occurs in 20% to 25% of cases and is associated with poor prognosis. Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a monoclonal antibody targeting the HER2 extracellular domain that has been shown to significantly reduce relapse rates. However, some patients with HER2+ tumors do not respond to Herceptin, and 60% to 85% of patients with HER2+ metastatic breast cancer acquire resistance within a short time period. In this review, we discuss proposed mechanisms of action of trastuzumab and trastuzumab resistance and various drugs that have been developed to overcome drug resistance. We introduce the basal molecular subtype as a predictor of increased risk in HER2+ breast cancer and a possible alternative cause of drug resistance.

  11. Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

    DEFF Research Database (Denmark)

    Nielsen, C U; Amstrup, J; Steffansen, B

    2001-01-01

    (max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P PepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control......The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis...... of the fluxes showed that maximal flux (V(max)) of transepithelial transport decreased from 3.00 +/- 0.17 nmol x cm(-2) x min(-1) in control cells to 0.50 +/- 0.07 nmol x cm(-2) x min(-1) in cells treated with 5 ng/ml EGF (n = 6, P

  12. Advances in Variations of Estrogen Receptor, Progesterone Receptor and Human Epidermal Growth Factor Receptor-2 Status in Metastatic Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Yuan Yuan; Zhang Lili

    2013-01-01

    Chemotherapy, endocrine therapy and molecular targeted therapy are vital means in the treatment of metastatic breast cancer (MBC), whose reasonable and standard applications are of great importance to prolong patients’ survival and improve the quality of life. The expressions of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) present signiifcant differences between primary and metastatic breast cancer. However, these differences may affect the selection of MBC patients for therapeutic strategies and judgment on the prognosis. Hence, the relevant researches on variations of hormone receptors and HER-2 in primary and metastatic breast cancer, discordant causes of ER, PR and HER-2 expression in primary and metastatic lesions and clinical value of biopsy to the metastases are reviewed in the study.

  13. Harnessing Integrative Omics to Facilitate Molecular Imaging of the Human Epidermal Growth Factor Receptor Family for Precision Medicine.

    Science.gov (United States)

    Pool, Martin; de Boer, H Rudolf; Hooge, Marjolijn N Lub-de; van Vugt, Marcel A T M; de Vries, Elisabeth G E

    2017-01-01

    Cancer is a growing problem worldwide. The cause of death in cancer patients is often due to treatment-resistant metastatic disease. Many molecularly targeted anticancer drugs have been developed against 'oncogenic driver' pathways. However, these treatments are usually only effective in properly selected patients. Resistance to molecularly targeted drugs through selective pressure on acquired mutations or molecular rewiring can hinder their effectiveness. This review summarizes how molecular imaging techniques can potentially facilitate the optimal implementation of targeted agents. Using the human epidermal growth factor receptor (HER) family as a model in (pre)clinical studies, we illustrate how molecular imaging may be employed to characterize whole body target expression as well as monitor drug effectiveness and the emergence of tumor resistance. We further discuss how an integrative omics discovery platform could guide the selection of 'effect sensors' - new molecular imaging targets - which are dynamic markers that indicate treatment effectiveness or resistance.

  14. Effects of recombinant human basic fibroblast growth factor on cell proliferation during mandibular fracture healing in rabbits

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To investigate the effects of recombinant human basicfibroblast growth factor (rhbFGF) on the cell proliferation during mandibular fracture healing in rabbits.Methods: The complex of rhbFGF and bovine type I collagen was implanted into the mandibular fracture site under periosteum of the animal. The whole mandible was harvested at 7, 14, 28, 56 and 84 days respectively after operation. The expression of proliferating cell nuclear antigen (PCNA) in callus was examined with immunohistochemical staining.Results: PCNA-positive cells in callus in the rhbFGF-treated group on days 7 and 14 were more than that in the control group (P<0.01).Conclusions: It indicates that rhbFGF can stimulate cell proliferation during mandibular fracture healing in rabbits.

  15. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    Science.gov (United States)

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  16. Exercise but not prostanoids enhance levels of vascular endothelial growth factor and other proliferative agents in human skeletal muscle interstitium

    DEFF Research Database (Denmark)

    Höffner, Lotte; Nielsen, Jens Jung; Langberg, Henning

    2003-01-01

    In the present study we examined whether exercise and prostanoids have an effect on the muscle interstitial concentration of vascular endothelial growth factor (VEGF) and on the proliferative effect of muscle interstitial fluid. Dialysate from resting and exercising human skeletal muscle, obtained...... either during control conditions or during cyclooxygenase inhibition, was examined for its content of VEGF and for its effect on endothelial cell proliferation. Microdialysis probes with high (960 kDa) and low (5 kDa) molecular-mass cut-off membranes were placed in the vastus lateralis muscle of healthy......Da dialysate from resting muscle than with perfusate and was 5.8-fold higher (P muscle. VEGF was not enhanced with exercise in the 5 kDa dialysate, yet the exercise dialysate induced a 1.9-fold higher (P

  17. Arecoline increases basic fibroblast growth factor but reduces expression of IL-1, IL-6, G-CSF and GM-CSF in human umbilical vein endothelium.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Moore, Malcolm A S; Zoellner, Hans

    2015-09-01

    Areca nut chewing is associated with oral submucous fibrosis (OSF). Raised vascular basic fibroblast growth factor may induce fibrosis. Arecoline is a muscarinic alkaloid in areca nut, which we earlier reported causes injury and necrosis of human endothelium. Human umbilical vein endothelial cells were exposed to arecoline with or without tumor necrosis factor-α, and separately to acetylcholine, muscarine, or nicotine. Protein levels of basic fibroblast growth factor, as well as the inflammatory cytokines: granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor, and Interleukins-6, 1-α and 1-β, were determined by enzyme-linked immunosorbent assay. mRNA levels were established by real-time reverse transcription polymerase chain reaction. Basic fibroblast growth factor was released into the culture medium at arecoline levels causing necrosis (P arecoline on levels of the inflammatory cytokines (P arecoline reduced this stimulated expression (P Arecoline had no effect on mRNA for basic fibroblast growth factor, although there was reduced mRNA for the separate inflammatory cytokines studied. The effect of acetylcholine, muscarine, and nicotine was minimal and dissimilar to that of arecoline. Data raise the possibility that arecoline-induced, vascular basic fibroblast growth factor contributes to OSF, by combining increased growth factor expression with endothelial necrosis, and thus driving fibroblast proliferation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias

    Energy Technology Data Exchange (ETDEWEB)

    Huebner, K.; Isobe, M.; Chao, M.; Bothwell, M.; Ross, A.H.; Finan, J.; Hoxie, J.A.; Sehgal, A.; Buck, C.R.; Lanahan, A.

    1986-03-01

    Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differential acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.

  19. Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity

    Institute of Scientific and Technical Information of China (English)

    Yu Tian; Ren-Yu Ding; Ying-Hui Zhi; Ren-Xuan Guo; Shuo-Dong Wu

    2006-01-01

    AIM: To examine the expression of p53 and vascular endothelial growth factor (VEGF) as well as microvessel count (MVC) and to investigate the role of VEGF as an angiogenic marker and the possible role of p53 in the regulation of angiogenesis in human gallbladder carcinoma.METHODS: Surgically resected specimens of 49 gallbladder carcinomas were studied by immunohistochemical staining for p53 protein, VEGF, and factor Ⅷ-related antigen. VEGF expression and mutant p53 expression were then correlated with Nevin stage,differentiation grade, MVC, and lymph node metastasis.RESULTS: Positive p53 protein and VEGF expressions were found in 61.2% and 63.3% of tumors, respectively.p53 and VEGF staining status was identical in 55.1%of tumors. The Nevin staging of p53- or VEGF-positive tumors was significantly later than that of negative tumors. The MVC in p53- or VEGF-positive tumors was significantly higher than that in negative tumors,and MVC in both p53- and VEGF-negative tumors was significantly lower than that in the other subgroups.CONCLUSION: Our findings suggest that p53-VEGF pathway can regulate tumor angiogenesis in human gallbladder carcinoma. Combined analysis of p53 and VEGF expression might be useful for predicting the tumor vascularity of gallbladder cancer.

  20. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  1. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol

    DEFF Research Database (Denmark)

    Kassem, M; Kveiborg, Marie; Eriksen, E F

    2000-01-01

    Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However, few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-...

  2. Inhibition of connective tissue growth factor attenuates paraquat-induced lung fibrosis in a human MRC-5 cell line.

    Science.gov (United States)

    Huang, Min; Yang, Huifang; Zhu, Lingqin; Li, Honghui; Zhou, Jian; Zhou, Zhijun

    2016-11-01

    Chronic exposure to Paraquat (PQ) may result in progressive pulmonary fibrosis and subsequent chronic obstructive pulmonary malfunction. Connective tissue growth factor (CTGF) has been proposed as a key determinant in the development of lung fibrosis. We investigated thus whether knock down of CTGF can prevent human lung fibroblasts (MRC-5) activation and proliferation with the subsequent inhibition of PQ-induced fibrosis. MRC-5 was transfected with CTGF-siRNAs and exposed to different concentrations of PQ. The siRNA-silencing efficacy was evaluated using western blotting analyses, qRT-PCR and flow cytometry. Next, the viability and migration of MRC-5 was determined. MMP-2, MMP-9, and TIMP-1 accumulation were quantified to evaluate the lung fibrosis exposure to PQ. Over expression of CTGF mRNA was observed in human MRC-5 cell as early as 6 h following PQ stimulation. CTGF gene expression in MRC-5 cells was substantially reduced by RNAi, which significantly suppressed the expression of the lung fibrosis markers such as tissue inhibitor of metalloproteinase-2 (TIMP-2), Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) that were stimulated by PQ. Inhibition of CTGF expression suppressed impeded the proliferation and migration ability of MRC-5 cells and resulted in cell-extracellular matrix (ECM) protein accumulation in cells. Our results suggest that CTGF promoted the development of PQ-induced lung fibrosis in collaboration with transforming growth factor β1 (TGFβ1). Furthermore, the observed arresting effects of CTGF knock down during this process suggested that CTGF is the potential target site for preventing PQ-induced pulmonary fibrosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1620-1626, 2016.

  3. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Sik-Loo Tan

    Full Text Available To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs using growth differentiation factor 5 (GDF5 was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome were significantly altered during the tenogenic differentiation process (corrected p<0.05. The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell

  4. Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, F.; Saya, H.; Bruner, J.M.; Morrison, R.S. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States))

    1994-01-18

    Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, the authors demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1[beta]) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1[alpha]) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1[alpha] to FGFR-2 and a shift in expression from FGFR-1[alpha] to FGFR-1[beta] as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.

  5. 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

    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...

  6. Impact of Growth Factor Independence 1 in Human T-Cell Lymphomas

    DEFF Research Database (Denmark)

    Dabrowska, Magdalena Julia; Dybkær, Karen; Johansen, Preben

    2009-01-01

    in the Gfi1 3'UTR have been demonstrated to decouple microRNA-mediated posttranscriptional regulation of protein expression (Dabrowska et al, 2009) further supporting its role in lymphomagenesis. In human cancers, Gfi1 protein expression has been observed in HTLV-1 induced ATLL and SCLC but no knowledge...

  7. Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-KB in human dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Dong-Wook HAN; Mi Hee LEE; Hak Hee KIM; Suong-Hyu HYON; Jong-Chul PARK

    2011-01-01

    Aim: To investigate the effects of (-)epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, on cell growth, cell cycle and phosphorylated nuclear factor-kB (pNF-KB) expression in neonatal human dermal fibroblasts (nHDFs).Methods: The proliferation and cell-cycle of nHDFs were determined using WST-8 cell growth assay and flow cytometry, respectively. The apoptosis was examined using DNA ladder and Annexin V-FITC assays. The expression levels of pNF-kB and cell cycle-related genes and proteins in nHDFs were measured using cDNA microarray analyses and Western blot. The cellular uptake of EGCG was examined using fluorescence (FITC)-Iabeled EGCG (FITC-EGCG) in combination with confocal microscopy.Results: The effect of EGCG on the growth of nHDFs depended on the concentration tested. At a low concentration (200 μmol/L), EGCG resulted in a slight decrease in the proportion of ceils in the S and G/M phases of cell cycle with a concomitant increase in the proportion of cells in G/G phase. At the higher doses (400 and 800 pmol/L), apoptosis was induced. The regulation of EGCG on the expression of pNF-kB was also concentration-dependent, whereas it did not affect the unphosphorylated NF-kB expression, cDNA microarray analysis showed that cell cycle-related genes were down-regulated by EGCG (200 μmol/L). The expression of cyclins A/B and cyclin-dependent kinase 1 was reversibly regulated by EGCG (200 μmol/L). FITC-EGCG was found to be internalized into the cyto-plasm and translocated into the nucleus of nHDFs.Conclusion: EGCG, through uptake into cytoplasm, reversibly regulated the cell growth and expression of cell cycle-related proteins and genes in normal fibroblasts.

  8. High molecular weight fibroblast growth factor-2 in the human heart is a potential target for prevention of cardiac remodeling.

    Directory of Open Access Journals (Sweden)

    Jon-Jon Santiago

    Full Text Available Fibroblast growth factor 2 (FGF-2 is a multifunctional protein synthesized as high (Hi- and low (Lo- molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD and 68% (±25 SD of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-AbHi-FGF-2 reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-AbHi-FGF-2 in vitro. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes

  9. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Mortazavi Yousef

    2016-07-01

    Full Text Available Objective Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF and assessed their neural lineage genes. Materials and Methods In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293 packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR. Results We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF, glial fibrillary acidic protein (GFAP and Microtubule-associated protein 2 (MAP2 genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF genes reduced. Conclusion Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this

  10. Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3.

    Science.gov (United States)

    Singh, Rana P; Tyagi, Anil K; Dhanalakshmi, Sivanandhan; Agarwal, Rajesh; Agarwal, Chapla

    2004-02-20

    Dietary intake of many fruits and vegetables has been shown to be associated with reduced risk of cancer. We investigated the in vivo efficacy of grape seed extract (GSE, patented as Traconol) against prostate cancer (PCA) and associated molecular events. Athymic nude mice were implanted with hormone-refractory human prostate carcinoma DU145 cells and fed with 100 and 200 mg/kg/day (5 days/week) doses of GSE for 7 weeks. At the end of experiment, tumors were immunohistochemically analyzed for cell proliferation, apoptosis and angiogenesis. Our data show that GSE feeding strongly inhibited tumor growth that accounted for 59-73% (p < 0.001) inhibition in tumor volume and 37-47% (p < 0.05) decrease in tumor weight at the end of the experiment. It did not show any significant change in body weight gain profile and diet consumption. Immunohistochemical analysis of tumors showed that GSE decreases proliferation index by 51-66% (p < 0.001) and increases apoptotic index by 3-4-fold (p < 0.001). CD31 staining for endothelial cells, showed decrease in intratumoral microvasculature in GSE-fed group of mice. Control tumors showed 64.0 +/- 1.6 CD31 positive cells/400x field compared to 23.2 +/- 0.9 and 15.7 +/- 0.08 (p < 0.001) CD31 positive cells in 100 and 200 mg/kg doses of GSE-treated tumors, respectively. GSE strongly inhibited (47-70%, p < 0.05) vascular endothelial growth factor (VEGF) secretion in conditioned medium by DU145 cells. Recently, the circulating level of insulin-like growth factor binding protein (IGFBP)-3 is shown to inversely related with PCA risk, growth and prognosis. Consistent with this, we observed 6-7-fold (p < 0.001) increase in tumor-secreted levels of IGFBP-3 after GSE feeding. In other immunohistochemical studies, compared to controls, tumor xenografts from GSE-fed groups of mice showed a moderate decrease in VEGF but an increase in IGFBP-3 levels. These findings suggest that GSE possesses in vivo anticancer efficacy against hormone

  11. The interaction between epidermal growth factor (EGF) and matrix metalloproteinase induces the development of sweat glands in human fetal skin

    Institute of Scientific and Technical Information of China (English)

    Li Jianfu; Fu Xiaobing; Sheng Zhiyong

    2001-01-01

    Objective:The development of sweat glands is a very complicated biological process involving many factors. In this study, we explore the inter-relationship between epidermal growth factor (EGF),matrix metalloproteinases (MMP-2,MMP-7) and development of sweat glands in human embryos. Furthermore, we hope to elucidate the mechanism(s) underlying the induction of epidermal stem cells into sweat gland cells. Methods:Skin biospies of human embryos obtained from spontaneous abortions at different gestational ages from 11 to 31 weeks were used in this study. The dynamical expression of EGF, MMP-2, MMP-7 and keratin-7 (K7) in developing sweat gland cells or extracellular stroma surrounding the sweat gland cells were examined with S-P immunohistochemical methods.The localization of the cellular sources of MMP-2 and MMP 7 was examined with in situ hybridization. Results:At 14-20 wk of gestation, a gradual increase in EGF immunoreactivity was observed not only in developing sweat gland buds but also in extracellular stroma surrounding the buds,and the expression intensity peaked at 20-22 wk of gesta- tional age. All mRNA-positive buds or cells in developing sweat glands contained corresponding immunoreactive proteins. Positive immunostaining for K7 appeared in early sweat gland buds at 14-16wk of gestation, and from then on, K7 was concentrated in developing sweat gland cords or cells. Conclusions: The morphogenesis of sweat gland in human fetal skin begins at 14-16wk of gestational age, and essentially completes by 24wk. There is a close relationship among EGF,extracellular matrix remodeling and morphogenesis of sweat glands, and EGF is one of the inducers in the development and maturity of sweat gland buds or cells.

  12. Secretory expression and characterization of a recombinant deleted variant of human hepatocyte growth factor in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Zhi-Min Liu; Hong-Liang Zhao; Chong Xue; Bing-Bing Deng; Wei Zhang; Xiang-Hua Xiong; Bing-Fen Yang; Xue-Qin Yao

    2005-01-01

    AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris.METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coliyeast shuttle vector of pPIC9. The constructed plasmid,pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut+ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor.RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

  13. Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this study the w/o/w extraction–evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.

  14. Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

    Institute of Scientific and Technical Information of China (English)

    DONG XiaoQing; XU Jun; WANG WeiCai; Luo Hao; LIANG XiaoFei; Zhang Lei; Wang HanJie; Wang PengHua; CHANG Jin

    2008-01-01

    In this study the w/o/w extraction-evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-Ioaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-ioaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.

  15. THE INVESTMENT IN HUMAN CAPITAL, AN INTRISIC FACTOR OF THE SUSTAINABLE ECONOMIC GROWTH

    Directory of Open Access Journals (Sweden)

    CRISTINA TEODORA BALACEANU

    2011-04-01

    Full Text Available The educational system will need to direct its actions and programs towards the identification of the current and future values of the labour market, starting from the existing and potential labour resources, anticipating first and foremost the adjusting of the economy to fast-developing fields and domains, put forward by the State via the Fast-developing Field Strategies or even via the Fast-developing National Strategy. It will accordingly generate a binder between the demands of the labour market as a response to the developing necessities of the economy, and the training/specialization of the labour force as offered by the national syllabus. By these means the educational system would create a labour force compatible with the labour market, which is both a premiss for the increasing level of employment and for the sustainable economic growth. Our task is therefore to provide a concept of education related to technological progress, based on the model of Nelson and Phelps, and a suggestion for investments and education policies.

  16. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-05-06

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of /sup 125/I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of /sup 125/I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of /sup 125/I-labeled human FSH to testis receptor.

  17. In vitro hepatic metabolism of cediranib, a potent vascular endothelial growth factor tyrosine kinase inhibitor: interspecies comparison and human enzymology.

    Science.gov (United States)

    Schulz-Utermoehl, Timothy; Spear, Michael; Pollard, Christopher R J; Pattison, Christine; Rollison, Helen; Sarda, Sunil; Ward, Michelle; Bushby, Nick; Jordan, Angela; Harrison, Mike

    2010-10-01

    The in vitro metabolism of cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors in late-stage development for the treatment of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from preclinical species and humans using radiolabeled material. In human hepatocyte cultures, oxidative and conjugative metabolic pathways were identified, with pyrrolidine N(+)-glucuronidation being the major route. The primary oxidative pathways were di-and trioxidations and pyrrolidine N-oxidation. All metabolites with the exception of the N(+)-glucuronide metabolite were observed in rat and cynomolgus monkey hepatocyte preparations. Additional metabolism studies in liver microsomes from these or other preclinical species (CD-1 mouse, Han Wistar rat, Dunkin Hartley guinea pig, Göttingen mini-pig, New Zealand White rabbit, beagle dog, and cynomolgus and rhesus monkey) indicated that the N(+)-glucuronide metabolite was not formed in these additional species. Incubations with recombinant flavin-containing monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) enzymes and inhibition studies using the nonselective cytochrome P450 (P450) chemical inhibitor 1-aminobenzotriazole in human hepatocytes indicated that FMO1 and FMO3 contributed to cediranib N-oxidation, whereas UGT1A4 had a major role in cediranib N(+)-glucuronidation. P450 enzymes had only a minor role in the metabolism of cediranib. In conclusion, species differences in the formation of the N(+)-glucuronide metabolite of cediranib were observed. All other metabolites of cediranib found in humans were also detected in rat and cynomolgus monkey. Non-P450 enzymes are predominantly involved in the metabolism of cediranib, and this suggests that clinical drug interactions involving other coadministered drugs are unlikely.

  18. The Mu opioid receptor promotes opioid and growth factor-induced proliferation, migration and Epithelial Mesenchymal Transition (EMT in human lung cancer.

    Directory of Open Access Journals (Sweden)

    Frances E Lennon

    Full Text Available Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05. Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

  19. Generation and characterization of a panel of monoclonal antibodies specific for human fibroblast growth factor receptor 4 (FGFR4).

    Science.gov (United States)

    Chen, Chaoyuan; Patel, Sima; Corisdeo, Susanne; Liu, Xiangdong; Micolochick, Holly; Xue, Jiyang; Yang, Qifeng; Lei, Ying; Wang, Baiyang; Soltis, Daniel

    2005-06-01

    Fibroblast growth factor receptor 4 (FGFR4) is a member of the FGFR family of receptor tyrosine kinases, and plays important roles in a variety of biological functions such as cell proliferation, differentiation, migration, angiogenesis, tissue repair, and tumorigenesis. The human FGFRs share a high degree of sequence homology between themselves, as well as with their murine homologs. Consequently, it has been suggested that it may be difficult to prepare monoclonal antibodies (MAbs) that are specific for the individual receptor types. In this communication, we report on the development and characterization of a panel of anti-human FGFR4 MAbs that were generated in mice using a rapid immunization protocol. Using a modified rapid immunization at multiple sites (RIMMS) protocol with the soluble extracellular domain of human FGFR4 (FGFR4-ECD), the immunized mice developed high levels of polyclonal IgG to the immunogen within 13 days of the first immunization. The lymph node cells isolated from the immunized animals were then fused with mouse myeloma cells for hybridoma generation. Use of an efficient hybridoma cloning protocol in combination with an ELISA screening procedure allowed for early identification of stable hybridomas secreting antihuman FGFR4 IgG. Several identified MAbs specifically reacted with the FGFR4 protein without binding to the other human isoforms (FGFR1, FGFR2, and FGFR3). As evaluated by BIAcore analysis, most anti-FGFR4 MAbs displayed high affinities (8.6 x 10(8) approximately 3.9 x 10(10) M) to FGFR4. Furthermore, these MAbs were able to bind to FGFR4 expressed on human breast tumor cell lines MDA-MB-361 and MDA-MB-453. Taken together, the results demonstrate that the RIMMS strategy is an effective approach for generating class-switched, high-affinity MAbs in mice to evolutionarily conserved proteins such as human FGFR4. These MAbs may be useful tools for further investigation of the biological functions and pathological roles of human FGFR4.

  20. Limited efficacy of COX-2 inhibitors on nerve growth factor and metalloproteinases expressions in human synovial fibroblasts.

    Science.gov (United States)

    Yorifuji, Makiko; Sawaji, Yasunobu; Endo, Kenji; Kosaka, Taiichi; Yamamoto, Kengo

    2016-05-01

    Nerve growth factor (NGF) is associated with arthritic pain and metalloproteinases are implicated in collagen and aggrecan degradation. Although selective COX-2 inhibitors are recommended for the treatment of arthritic diseases, their effects on NGF and metalloproteinases remain unclear. This study investigated the regulations of NGF and metalloproteinases by selective COX-2 inhibitors in isolated human synovial cells. The isolated human synovial cells were stimulated with IL-1β in the presence of selective COX-2 inhibitors (NS-398 or celecoxib) with or without exogenous PGE2 or its receptor (EP1-4) agonists. The expressions of NGF, MMP-1, -3, -13, ADAMTS-4, and -5 were quantified by real-time PCR and their proteins were determined by Western blotting. The amount of PGE2 released was measured by enzyme-linked immunosorbent assay (ELISA). The IL-1β inductions of NGF and MMP-1 and MMP-13 were augmented by the COX-2 inhibitors, whereas the inductions of ADAMTS-4 and ADAMTS-5 were inhibited. These actions were reversed by supplementing PGE2 or the EP4 agonist exogenously. Our comprehensive analysis revealed that COX-2 inhibitors may be beneficial for suppressing aggrecan degradation and for reducing inflammatory pain by inhibiting PGE2 release, although they may have limited efficacy in suppressing collagen degradation and nerve growth. This study suggests the feedback roles of PGE2 in the negative regulation of NGF and MMP-1 and MMP-13 and the positive regulation of ADAMTS-4 and ADAMTS-5. Copyright © 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.

  1. Pretreatment with recombinant human vascular endothelial growth factor virus replication and inflammation in a perinatal lamb model of RSV infection

    Science.gov (United States)

    Vascular endothelial growth factor (VEGF) is increasingly recognized as a perinatal regulator of lung maturation and surfactant protein expression. Innate immune components including surfactant proteins A and D, and beta defensins have putative antimicrobial activity against pulmonary pathogens inc...

  2. The effect of passive movement training on angiogenic factors and capillary growth in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Rufener, Nora; Bojsen-Møller, Jens

    2010-01-01

    Abstract The effect of a period of passive movement training on angiogenic factors and capillarization in skeletal muscle was examined. Seven young males were subjected to passive training for 90 min, four times/week in a motor-driven knee extensor device that extended one knee passively at 80...... cycles/min. The other leg was used as control. Muscle biopsies were obtained from m. v. lateralis of both legs before as well as after 2 and 4 weeks of training. After the training period, passive movement and active exercise were performed with both legs and muscle interstitial fluid was sampled from...... legs. Acute passive movement increased (P muscle interstitial VEGF levels 4-6 -fold above rest and the proliferative effect, determined in vitro, of the muscle interstitial fluid ~16-fold compared to perfusate. These increases were similar for active exercise. The results demonstrate...

  3. Antisense oligonucleotide to insulin—like growth factor Ⅱ induces apotosis in human ovarian cancer AO cell line

    Institute of Scientific and Technical Information of China (English)

    YINDELING; LUPU; 等

    1998-01-01

    The effects of antisense oligonucleotide to insulin0like growth factor -Ⅱ(IGFⅡ)to induce apotosis in human ovarian cancer cells were evaluated.Antiproliferation effects of antisense to IGFⅡin ovarian cancer AO cells were determined by 3H-thymidine incorporation.Apoptosis of the IGFⅡ antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide,IGFⅡ antisense(4.5μM) treatment of 48h maximally inhibited proliferation of AO cells,More than 25% of IGFⅡantisense-treated cells(4.5μM for 24h) had undergone apoptosis,whereas less than 3% of the cells were apoptotic in either IGFⅡ sense-treated cells or untreated cells.Antisense oligonucleotide to IGFⅡ significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell.These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFⅡ may serve as a therapeutic approach.

  4. Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

    Institute of Scientific and Technical Information of China (English)

    SUNBAODONG; JINDANSONG

    1995-01-01

    Human amniotic basement membrane (HABM) model and agarose drop explant method were used to investigate the effects of retinoic acid(RA) on the invasive ness and adhesiveness to the basement membrane,and the migration of a highly invasive human colorectal cancer cell line CCL229.Results showed that 5×106 MRA markedly reduced the in vitro invasiveness and adhesiveness to the HABM,and the migration of the CCL229 cells.In addition,to elucidate the relation between expression of epidermal growth factor receptor(EGFR) and the invasiveness of the colorectal carcinoma cells,two well-differentiated,but with different invasiveness colorectal cancer cell lines were compared at mRNA level for expression of EGFR by using EGFR cDNA probe labeled with digoxigenin(DIG). Expression of EGFR was shown to be markedly higher in the highly invassive CCL229 cells than that in the low invasive CX-1 cells.Furthermore,expression of EGFR in RA treated CCL229 cells gradually decreased with time,the level being the lowest on day 6 of the RA treatment.

  5. Expression of nerve growth factor (NGF, TrkA and p75NTR in developing human foetal teeth

    Directory of Open Access Journals (Sweden)

    Thimios A. Mitsiadis

    2016-08-01

    Full Text Available Nerve growth factor (NGF is important for the development and the differentiation of neuronal and non-neuronal cells. NGF binds to specific low- and high-affinity cell surface receptors, respectively p75NTR and TrkA. In the present study, we examined by immunohistochemistry the expression patterns of the NGF, p75NTR and TrkA proteins during human foetal tooth development, in order to better understand the mode of NGF signalling action in dental tissues. The results obtained show that these molecules are expressed in a wide range of dental cells of both epithelial and mesenchymal origin during early stages of odontogenesis, as well as in nerve fibres that surround the developing tooth germs. At more advanced developmental stages, NGF and TrkA are localised in differentiated cells with secretory capacities such as preameloblasts/ameloblasts secreting enamel matrix and odontoblasts secreting dentine matrix. In contrast, p75NTR expression is absent from these secretory cells and restricted in proliferating cells of the dental epithelium. The temporospatial distribution of NGF and p75NTR in foetal human teeth is similar, but not identical, with that observed previously in the developing rodent teeth, thus indicating that the genetic information is well conserved during evolution. The expression patterns of NGF, p75NTR and TrkA during odontogenesis suggest regulatory roles for NGF signalling in proliferation and differentiation of epithelial and mesenchymal cells, as well as in attraction and sprouting of nerve fibres within dental tissues.

  6. Expression of Nerve Growth Factor (NGF), TrkA, and p75NTR in Developing Human Fetal Teeth

    Science.gov (United States)

    Mitsiadis, Thimios A.; Pagella, Pierfrancesco

    2016-01-01

    Nerve growth factor (NGF) is important for the development and the differentiation of neuronal and non-neuronal cells. NGF binds to specific low- and high-affinity cell surface receptors, respectively, p75NTR and TrkA. In the present study, we examined by immunohistochemistry the expression patterns of the NGF, p75NTR, and TrkA proteins during human fetal tooth development, in order to better understand the mode of NGF signaling action in dental tissues. The results obtained show that these molecules are expressed in a wide range of dental cells of both epithelial and mesenchymal origin during early stages of odontogenesis, as well as in nerve fibers that surround the developing tooth germs. At more advanced developmental stages, NGF and TrkA are localized in differentiated cells with secretory capacities such as preameloblasts/ameloblasts secreting enamel matrix and odontoblasts secreting dentine matrix. In contrast, p75NTR expression is absent from these secretory cells and restricted in proliferating cells of the dental epithelium. The temporospatial distribution of NGF and p75NTR in fetal human teeth is similar, but not identical, with that observed previously in the developing rodent teeth, thus indicating that the genetic information is well-conserved during evolution. The expression patterns of NGF, p75NTR, and TrkA during odontogenesis suggest regulatory roles for NGF signaling in proliferation and differentiation of epithelial and mesenchymal cells, as well as in attraction and sprouting of nerve fibers within dental tissues. PMID:27536251

  7. Interaction between Fibrinogen and Insulin-Like Growth Factor-Binding Protein-1 in Human Plasma under Physiological Conditions.

    Science.gov (United States)

    Gligorijević, N; Nedić, O

    2016-02-01

    Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.

  8. Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta.

    Science.gov (United States)

    Watson, Chris J; Phelan, Dermot; Collier, Patrick; Horgan, Stephen; Glezeva, Nadia; Cooke, Gordon; Xu, Maojia; Ledwidge, Mark; McDonald, Kenneth; Baugh, John A

    2014-06-01

    Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

  9. Deregulation of the Pit-1 transcription factor in human breast cancer cells promotes tumor growth and metastasis

    Science.gov (United States)

    Ben-Batalla, Isabel; Seoane, Samuel; Garcia-Caballero, Tomas; Gallego, Rosalia; Macia, Manuel; Gonzalez, Luis O.; Vizoso, Francisco; Perez-Fernandez, Roman

    2010-01-01

    The Pit-1 transcription factor (also know as POU1F1) plays a critical role in cell differentiation during organogenesis of the anterior pituitary in mammals and is a transcriptional activator for pituitary gene transcription. Increased expression of Pit-1 has been reported in human tumorigenic breast cells. Here, we found that Pit-1 overexpression or knockdown in human breast cancer cell lines induced profound phenotypic changes in the expression of proteins involved in cell proliferation, apoptosis, and invasion. Some of these protumorigenic effects of Pit-1 were mediated by upregulation of Snai1, an inductor of the epithelial-mesenchymal transition. In immunodeficient mice, Pit-1 overexpression induced tumoral growth and promoted metastasis in lung. In patients with invasive ductal carcinoma of the breast and node-positive tumor, high expression of Pit-1 was significantly correlated with Snai1 positivity. Notably, in these patients elevated expression of Pit-1 was significantly and independently associated with the occurrence of distant metastasis. These findings suggest that Pit-1 could help to make a more accurate prognosis in patients with node-positive breast cancer and may represent a new therapeutic target. PMID:21060149

  10. ISS Payload Human Factors

    Science.gov (United States)

    Ellenberger, Richard; Duvall, Laura; Dory, Jonathan

    2016-01-01

    The ISS Payload Human Factors Implementation Team (HFIT) is the Payload Developer's resource for Human Factors. HFIT is the interface between Payload Developers and ISS Payload Human Factors requirements in SSP 57000. ? HFIT provides recommendations on how to meet the Human Factors requirements and guidelines early in the design process. HFIT coordinates with the Payload Developer and Astronaut Office to find low cost solutions to Human Factors challenges for hardware operability issues.

  11. Fibroblast growth factor-23 induces cellular senescence in human mesenchymal stem cells from skeletal muscle.

    Science.gov (United States)

    Sato, Chisato; Iso, Yoshitaka; Mizukami, Takuya; Otabe, Koji; Sasai, Masahiro; Kurata, Masaaki; Sanbe, Takeyuki; Sekiya, Ichiro; Miyazaki, Akira; Suzuki, Hiroshi

    2016-02-12

    Although muscle wasting and/or degeneration are prevalent in patients with chronic kidney disease, it remains unknown whether FGF-23 influences muscle homeostasis and regeneration. Mesenchymal stem cells (MSCs) in skeletal muscle are distinct from satellite cells and have a known association with muscle degeneration. In this study we sought to investigate the effects of FGF-23 on MSCs isolated from human skeletal muscle in vitro. The MSCs expressed FGF receptors (1 through 4) and angiotensin-II type 1 receptor, but no traces of the Klotho gene were detected. MSCs and satellite cells were treated with FGF-23 and angiotensin-II for 48 h. Treatment with FGF-23 significantly decreased the number of MSCs compared to controls, while treatment with angiotensin-II did not. FGF-23 and angiotensin-II both left the cell counts of the satellite cells unchanged. The FGF-23-treated MSCs exhibited the senescent phenotype, as judged by senescence-associated β-galactosidase assay, cell morphology, and increased expression of p53 and p21 in western blot analysis. FGF-23 also significantly altered the gene expression of oxidative stress regulators in the cells. In conclusion, FGF-23 induced premature senescence in MSCs from skeletal muscle via the p53/p21/oxidative-stress pathway. The interaction between the MSCs and FGF-23 may play a key role in the impaired muscle reparative mechanisms of chronic kidney disease.

  12. Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer's Disease.

    Science.gov (United States)

    McGinley, Lisa M; Sims, Erika; Lunn, J Simon; Kashlan, Osama N; Chen, Kevin S; Bruno, Elizabeth S; Pacut, Crystal M; Hazel, Tom; Johe, Karl; Sakowski, Stacey A; Feldman, Eva L

    2016-03-01

    Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar "best in class" cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD. ©AlphaMed Press.

  13. Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

    Science.gov (United States)

    Wang, Qiaoyun; Dierkes, Rüdiger; Kaufmann, Rainer; Cremer, Christoph

    2014-04-01

    In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1 and 3 in Gastric and Esophageal Adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Charlotta Hedner

    Full Text Available Gastric and esophageal adenocarcinomas are major global cancer burdens. These cancer forms are characterized by a poor prognosis and a modest response to chemo- radio- and targeted treatment. Hence there is an obvious need for further enhanced diagnostic and treatment strategies. The aim of this study was to examine the expression and prognostic impact of human epidermal growth factor receptor 1 (HER1/EGFR and 3 (HER3, as well as the occurrence of EGFR and KRAS mutations in gastric and esophageal adenocarcinoma.Immunohistochemical expression of EGFR and HER3 was analysed in all primary tumours and a subset of lymph node metastases in a consecutive cohort of 174 patients with adenocarcinoma of the stomach, cardia and esophagus. The anti-HER3 antibody used was validated by siRNA-mediated knockdown, immunohistochemistry and quantitative real-time PCR. EGFR and KRAS mutation status was analysed by pyrosequencing tecchnology.High EGFR expression was an independent risk factor for shorter overall survival (OS, whereas high HER3 expression was associated with a borderline significant trend towards a longer OS. KRAS mutations were present in only 4% of the tumours and had no prognostic impact. All tumours were EGFR wild-type. These findings contribute to the ongoing efforts to decide on the potential clinical value of different HERs and druggable mutations in gastric and esophageal adenocarcinomas, and attention is drawn to the need for more standardised investigational methods.

  15. Morphine induces expression of platelet-derived growth factor in human brain microvascular endothelial cells: implication for vascular permeability.

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

    Full Text Available Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling and loss-of-function (Egr-1 approaches demonstrated the role of mitogen-activated protein kinases (MAPKs, PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

  16. Effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6 in a serum-free medium.

    Directory of Open Access Journals (Sweden)

    Tokiwa,Takayoshi

    1989-12-01

    Full Text Available The effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6, which was inoculated at low density in a serum-free medium was examined. Several supplements were required to enhance cell growth of HuH-6. These included cholera toxin (CT, glucagon (Glu and selenium (Se. Type IV collagen (C-IV provided the most conductive environment tested for cell growth. These results suggest that CT, Glu, Se, and C-IV are important stimulators for the continuous growth of HuH-6 in a serum-free medium at low density.

  17. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  18. Human fibroblast growth factor 20 (FGF-20; CG53135-05): a novel cytoprotectant with radioprotective potential.

    Science.gov (United States)

    Maclachlan, T; Narayanan, B; Gerlach, V L; Smithson, G; Gerwien, R W; Folkerts, O; Fey, E G; Watkins, B; Seed, T; Alvarez, E

    2005-08-01

    The aim was to evaluate the radioprotective properties of recombinant human fibroblast growth factor 20 (FGF-20; CG53135-05) in vitro and in vivo and to examine its effects on known cellular pathways of radioprotection. Relative transcript levels of the cyclooxygenase 2 (COX2), Mn-super oxide dismutase (SOD), CuZn-SOD, extracellular (EC)-SOD, nuclear respiratory factor 2 (Nrf2), glutathione peroxidase 1 (GPX1) and intestinal trefoil factor 3 (ITF3) genes, which are involved in radiation response pathways, were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) in NIH/3T3, IEC18, CCD-18Co, CCD-1070sk and human umbilical vein endothelial cells (HUVEC) cells exposed to FGF-20. Activation of the radioprotective signal transduction pathways initiating with the serine/threonine Akt kinase and the extracellular regulated kinase (ERK) were analysed. Levels of intracellular hydrogen peroxide and cytosolic redox potential were also measured in irradiated and unirradiated cells in the presence or absence of FGF-20. The effects of FGF-20 on cell survival in vitro following ionizing radiation were evaluated using clonogenic assays. To test the potential activity of FGF-20 as a radioprotectant in vivo, mice were administered a single dose of FGF-20 (4 mg kg(-1), intraperitoneally (i.p.) 1 day before lethal total-body irradiation and evaluated for survival. In vitro exposure to FGF-20 increased expression of the Nrf2 transcription factor and oxygen radical scavenging enzymes such as MnSOD, activated signal transduction pathways (ERK and Akt) and resulted in increased survival of irradiated cells in vitro. FGF-20 treatment also resulted in a concomitant reduction in intracellular levels of injurious reactive oxygen species (ROS) following acute ionizing irradiation. Finally, prophylactic administration of FGF-20 to mice before potentially lethal, whole-body X-irradiation led to significant increases in overall survival. FGF-20 reduced the lethal effects of acute

  19. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaoou [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Liu, Lian [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Wen, Fuqiang, E-mail: wenfuqiang.scu@gmail.com [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China)

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

  20. Prediction of recurrence risk in early breast cancer using human epidermal growth factor 2 and cyclin A2

    Institute of Scientific and Technical Information of China (English)

    LI Hui-ping; XIAO Yu; WANG You-fan; L(U) You-yong; SUN Yan; JI Jia-fu; HOU Kuan-yong; LEI Yu-tao; ZHAO Hong-mei; WANG Jing; ZHENG Jie; LIU Jian-ying; WANG Mo-pei

    2010-01-01

    Background Human epidermal growth factor 2 (HER2) is one of the most important prediction factors, but only 25%-30% of breast cancer patients HER2 are positive. It is unknown whether there are other molecular markers that could be used to predict prognosis and recurrence in HER2 negative patients.This study investigated correlations of cyclin A2 and HER2 levels with clinical outcomes in 281 patients with invasive breast cancer in order to identify whether cyclin A2 can serve as a prognostic factor in HER2 negative patients.Methods Immunohistochemical staining was used to detect cyclin A2 and HER2 expression in 281 patients. Cyclin A2. and HER2 gene amplifications were analyzed using gene analysis and RT-PCR in 12 patients. Risk and survival estimates were analyzed using Log-rank, Kaplan-Meier, and Cox regression analysis; cyclin A2 and HER2 consistency with survival were analyzed using Kappa analysis.Results Patients with higher cyclin A2 and HER2 expressions had significantly shorter disease-free survival periods (P=0.047 and P=0.05, respectively). Kappa analysis performed that cyclin A2 and HER2 showed a low Kappa index (kappa=0.37), allowing us to conclude that cyclin A2 and HER2 detect different pathologies. Gene analysis and RT-PCR showed that cyclin A2 was upregulated in patients with early relapse; the average increase was 3.69-2.74 fold.Conclusions Cyclin A2 and HER2 are associated with proliferation and high recurrence, particularly when combined. Cyclin A2 is easily detected by nuclear staining and might be a useful biomarker for recurrence risk in HER2 negative patients.

  1. Association between cadmium and breast cancer risk according to estrogen receptor and human epidermal growth factor receptor 2: epidemiological evidence.

    Science.gov (United States)

    Strumylaite, Loreta; Kregzdyte, Rima; Bogusevicius, Algirdas; Poskiene, Lina; Baranauskiene, Dale; Pranys, Darius

    2014-05-01

    The study aimed to examine the association between cadmium (Cd) and the risk of breast cancer according to estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). A hospital-based case-control study was carried out in 585 cases and 1,170 controls. Information on possible risk factors was collected via a structured questionnaire. Urinary Cd was determined by atomic absorption spectrometry. The ER and HER2 levels in tumor tissue were analyzed by immunohistochemistry. Logistic regression was used to calculate odds ratios (ORs) and 95 % confidence intervals (CIs) for breast cancer by creatinine-adjusted urinary Cd. Women with greater creatinine-adjusted urine Cd (3rd quartile: 0.241-0.399 μg/g and 4th quartile: ≥ 0.4 μg/g) experienced 1.6 times higher risk of breast cancer compared with those having Cd concentration lower than 0.147 μg/g (1st quartile) [OR = 1.6, (95 % CI 1.19, 2.17) and OR = 1.62 (95 % CI 1.19, 2.21), respectively, P trend = 0.001] after adjustment for age and other confounders. Both ER+ and HER2- cases from the highest quartile of urine Cd exhibited approximately twice the breast cancer risk of those in the lowest quartile [OR = 1.9, (95 % CI 1.31, 2.74) and OR = 1.87, (95 % CI 1.33, 2.62), respectively, P trend cadmium as a risk factor for breast cancer, especially for both ER+ and HER2- cancer patients.

  2. Growth hormone, growth factors, and acromegaly

    Energy Technology Data Exchange (ETDEWEB)

    Ludecke, D.K.; Tolis, G.T.

    1987-01-01

    This book contains five sections, each consisting of several papers. The section headings are: Biochemistry and Physiology of GH and Growth Factors, Pathology of Acromegaly, Clinical Endocrinology of Acromegaly, Nonsurgical Therapy of Acromegaly, and Surgical Therapy of Acromegaly.

  3. A hepatoprotective Lindera obtusiloba extract suppresses growth and attenuates insulin like growth factor-1 receptor signaling and NF-kappaB activity in human liver cancer cell lines

    Directory of Open Access Journals (Sweden)

    Stroh Thorsten

    2011-05-01

    Full Text Available Abstract Background In traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC cell lines and the signaling pathways involved. Methods Four human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined. Results L.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase. Conclusions The traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary treatment option for HCC.

  4. Insulin-like growth factor binding protein 1 and human embryonic development during 6-10 gestational weeks

    Institute of Scientific and Technical Information of China (English)

    方群; 王艳霞; 周祎

    2004-01-01

    Background Insulin-like growth factor binding protein-1 (IGFBP-1), which is a carrier of Insulin-like growth factors (IGFs) regulates the fetal development by working as an active factor controlling the combination of IGFs with their receptors. This study was designed to investigate the relationship between IGFBP-1 and human embryonic development during weeks 6 -10 of gestation.Methods A total of 44 pregnant women with singleton pregnancy were divided into two groups: one with abnormal embryo development (n = 32) and the other with normal embryo development (n = 12).Enzyme-linked immunosorbent assay (ELISA) was employed to detect IGFBP-1 levels in maternal serum and decidual tissue. The expression of IGFBP-1 mRNA in deciduas was examined by reverse transcription polymerase chain reaction (RT-PCR) technique.Results The level of IGFBP-1 protein in maternal serum was significantly higher in the abnormal group [ (125.36 ± 47.93) μg/ml] than in the normal group [(70.72 ± 21.21) μg/ml ]. Both of IGFBP-1 and IGFBP-1 mRNA in deciduas were higher in abnormal group [ (1.60 ± 1.39) μg/ml and 1.66 ± 1.64, respectively ] than in the normal group [ (0.35 ± 0.23) μg/mi and 0.40 ± 0.20,respectively]. The level of IGFBP-1 in maternal serum was positively correlated with IGFBP-1 mRNA (r=0. 90, P<0.05) and IGFBP-1 protein (r=0.92, P<0.05) in decidual tissue.Conclusions During weeks 6 -10 of gestation, abnormal embryonic development is correlated with elevated IGFBP-1. The level of IGFBP-1 in maternal serum is related to the concentrations of IGFBP1 mRNA and IGFBP-1 in decidual tissue. The IGFBP-1 level in maternal serum may be used as a predictive marker to evaluate embryonic development.

  5. Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation.

    Science.gov (United States)

    Chang, Ki-Hwan; Kim, Min-Soo; Hong, Gwang-Won; Seo, Mi-Sun; Shin, Yong-Nam; Kim, Se-Ho

    2012-08-01

    It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the affinity of ER414. We obtained a 3D model of ER414 to identify the amino acids in the CDRs that needed to be mutated. Clones were selected from the phage library with randomized amino acids in the CDRs and substitution of amino acids in the HCDR3 and LCDR1 of ER414 led to improved affinity. A clone, H3-14, with a ~20-fold increased affinity, was selected from the HCDR3 randomized library. Then three clones, ER2, ER78 and ER79, were selected from the LCDR1 randomized library based on the H3-14 but did not show further increased affinities compared to that of H3-14. Of the three, ER2 was chosen for further characterization due to its better expression than others. We successfully performed affinity maturation of ER414 and obtained antibodies with a similar affinity as cetuximab. And antibody from an affinity maturation inhibits the EGF-mediated tyrosine phosphorylation of EGFR in a manner similar to cetuximab.

  6. Growth Factors for the Treatment of Ischemic Brain Injury (Growth Factor Treatment

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    Amara Larpthaveesarp

    2015-04-01

    Full Text Available In recent years, growth factor therapy has emerged as a potential treatment for ischemic brain injury. The efficacy of therapies that either directly introduce or stimulate local production of growth factors and their receptors in damaged brain tissue has been tested in a multitude of models for different Central Nervous System (CNS diseases. These growth factors include erythropoietin (EPO, vascular endothelial growth factor (VEGF, brain-derived neurotrophic factor (BDNF, and insulin-like growth factor (IGF-1, among others. Despite the promise shown in animal models, the particular growth factors that should be used to maximize both brain protection and repair, and the therapeutic critical period, are not well defined. We will review current pre-clinical and clinical evidence for growth factor therapies in treating different causes of brain injury, as well as issues to be addressed prior to application in humans.

  7. Growth factors for the treatment of ischemic brain injury (growth factor treatment).

    Science.gov (United States)

    Larpthaveesarp, Amara; Ferriero, Donna M; Gonzalez, Fernando F

    2015-04-30

    In recent years, growth factor therapy has emerged as a potential treatment for ischemic brain injury. The efficacy of therapies that either directly introduce or stimulate local production of growth factors and their receptors in damaged brain tissue has been tested in a multitude of models for different Central Nervous System (CNS) diseases. These growth factors include erythropoietin (EPO), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor (IGF-1), among others. Despite the promise shown in animal models, the particular growth factors that should be used to maximize both brain protection and repair, and the therapeutic critical period, are not well defined. We will review current pre-clinical and clinical evidence for growth factor therapies in treating different causes of brain injury, as well as issues to be addressed prior to application in humans.

  8. Novel Method of Cell-Free In Vitro Synthesis of the Human Fibroblast Growth Factor 1 Gene

    Directory of Open Access Journals (Sweden)

    Peijun Zuo

    2010-01-01

    Full Text Available Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based on Taq polymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1 gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1 the design of the DNA oligonucleotides to be assembled and (2 the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

  9. High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications.

    Science.gov (United States)

    Pizarro, Shelly A; Gunson, Jane; Field, Matthew J; Dinges, Rachel; Khoo, Stefanie; Dalal, Milind; Lee, Michael; Kaleas, Kimberly A; Moiseff, Kathryn; Garnick, Susan; Reilly, Dorothea E; Laird, Michael W; Schmelzer, Charles H

    2010-08-01

    Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells. Copyright 2010 Elsevier Inc. All rights reserved.

  10. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

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    V Chandana Epa

    Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  11. Association of human epidermal growth factor receptor 2 status with ipsilateral breast tumor recurrence and resistance to endocrine therapy

    Science.gov (United States)

    ISHITOBI, MAKOTO; SHIBA, MIZUHO; NAKAYAMA, TAKAHIRO; KOYAMA, HIROKI; TAMAKI, YASUHIRO

    2015-01-01

    The effect of prior endocrine therapy on tumor biology and clinical outcomes of locoregional recurrence remains unclear. A total of 76 patients, who underwent salvage breast surgery for estrogen receptor-positive ipsilateral breast tumor recurrence (IBTR) following breast-conserving surgery for primary breast cancer, were retrospectively reviewed to investigate the association of human epidermal growth factor receptor 2 (HER2) status with the characteristics of IBTR and clinical outcomes following resection of IBTR. There was a tendency for more patients with HER2-positive IBTR to show resistance to endocrine therapy (71.4%) compared with those with HER2-negative IBTR (37.3%) (P=0.085). Patients with HER2-positive IBTR exhibited a higher rate of recurrence (62.5%) compared with those with HER2-negative IBTR (25.0%) (P=0.027). These results suggested that HER2-positive IBTR may be associated with resistance to endocrine therapy and a poorer clinical outcome. PMID:26171193

  12. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

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    Balan Raluca

    2010-09-01

    Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

  13. Intranasal "painless" human Nerve Growth Factor [corrected] slows amyloid neurodegeneration and prevents memory deficits in App X PS1 mice.

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    Simona Capsoni

    Full Text Available Nerve Growth Factor (NGF is being considered as a therapeutic candidate for Alzheimer's disease (AD treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V, which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8, hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.

  14. GENISTEIN INHIBITS EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN HER-2/NEU TRANSFECTED HUMAN BREAST CANCER MCF-7 CELLS

    Institute of Scientific and Technical Information of China (English)

    ZHU Jun-dong; YU Xiao-ping; MI Man-tian

    2006-01-01

    Objective: our previous studies have demonstrated that HER-2/neu gene expression in human breast cancer MCF-7 cells promotes angiogenesis in MCF-7 cells xenograft tumors, and genistein inhibits angiogenesis in MCF-7 cells with HER-2/neu expression xenograft tumors. Here, the effects of genistein on the expression of vascular endothelial growth factor (VEGF) inMCR-7 cells with HER-2/neu expression were further studied for exploring the molecular mechanism of anti-angiogenesis in HER-2/neu-overexpressing breast cancer by genistein. Methods: HER-2/neu-overexpressing MCF-7 cells (MCF-7/HER-2)were established by transfecting HER-2/neu gene into HER-2/neu negative expression breast cancer MCF-7 cells.Immunocytochemical staining, western blot and reverse transcription-polymerase chain reaction (RT-PCR) were adopted to measure the expression of VEGF in MCF-7/HER-2 cells treated by genistein for 24, 48 and 72h. Results: HER-2/neu expression up-regulated VEGF mRNA and protein in MCF-7 cells, genistein decreased VEGF mRNA and protein level in MCF-7/HER-2 cells in a time-dependent manner. Conclusion: These results suggest that VEGF plays an important role in HER-2/neu gene expression promoted antiogenesis in breast cancer and genistein induced down-regulation of the expression of VEGF may be one of the molecular mechanisms of its anti-angiogenesis in HER-2/neu-overexpressing breast cancer.

  15. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

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    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  16. Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

    Science.gov (United States)

    Meng, Hsien-Wei; Pagano, John M; White, Brian S; Toyoda, Yoshiko; Min, Irene M; Craighead, Harold G; Shalloway, David; Lis, John T; Xiao, Kai; Jin, Moonsoo M

    2014-01-01

    SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (K(D)  = ∼ 1 nM) and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

  17. Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Hsien-Wei Meng

    Full Text Available SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (K(D  = ∼ 1 nM and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

  18. Mammographic features of calcifications in DCIS: correlation with oestrogen receptor and human epidermal growth factor receptor 2 status

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Min Sun; Moon, Woo Kyung; Chang, Jung Min; Cho, Nariya [Seoul National University College of Medicine, Department of Radiology, Seoul (Korea, Republic of); Park, So Yeon; Won, Jae-Kyung; Jeon, Yoon-Kyung; Park, In Ae [Seoul National University College of Medicine, Department of Pathology, Seoul (Korea, Republic of); Moon, Hyeong-Gon; Han, Wonshik [Seoul National University College of Medicine, Department of Surgery, Seoul (Korea, Republic of)

    2013-08-15

    This study investigated the correlation of oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status with the probability of malignancy (POM) of mammographic calcifications in ductal carcinoma in situ (DCIS). A total of 101 women (age range, 27-83 years) with pure DCIS that presented as mammographic calcifications were included. Three radiologists independently reviewed mammograms according to the BI-RADS lexicon and provided 100-point POM scores and a BI-RADS category. ER, HER2 and breast cancer subtypes were determined using immunohistochemistry (IHC) and fluorescence in situ hybridisation. Pairwise correlations between POM and IHC biomarker scores were calculated, and mammographic features were compared between breast cancer subtypes. HER2 level positively correlated with the POM score (P < 0.0001) and BI-RADS category (P < 0.0001), and ER level inversely correlated with the POM score (P < 0.013) and BI-RADS category (P < 0.010). Fine linear branching (P = 0.004) and segmental (P = 0.014) calcifications were significantly associated with HER2-positive cancers, and clustered calcifications were more frequently observed in ER-positive cancers (P = 0.014). HER2 status in DCIS correlated positively with the POM of mammographic calcifications, as determined by radiologists on the basis of the BI-RADS lexicon. (orig.)

  19. Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells.

    Science.gov (United States)

    Lo Vasco, Vincenza Rita; Leopizzi, Martina; Puggioni, Chiara; Della Rocca, Carlo; Businaro, Rita

    2014-03-01

    Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC γ1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3-6-h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.

  20. Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    RUI Yun-feng; DU Lin; WANG You; WANG Yang; LUI Pauline po-yee; TANG Ting-ting; CHAN Kai-ming; DAI Ke-rong

    2010-01-01

    Background Synovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type Ⅱ, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type Ⅱ, aggrecan, SOX9, link-protein, collagen type X and BMP receptor Ⅱ. Results Cells isolated under the optimized culturing density (104/60 cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99%) for CD44, CD90, CD105 and negative (<10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type Ⅱ was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type Ⅱ and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

  1. Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

    Science.gov (United States)

    Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua

    2015-08-01

    T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

  2. Expression of transforming growth factor-α and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

    Institute of Scientific and Technical Information of China (English)

    Jing Zhang; Wen-Liang Wang; Qing Li; Qing Qiao

    2004-01-01

    AIM: To evaluate the expression of transforming growth factor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance. METHODS: Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control. RESULTS: The TGF-α positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88. t%(52/59), respectively. TGF-α positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-α positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P<0.05). The TGF-α positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts). The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively. HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca. There was a prominent positive correlation between TGF-α and HBsAg expression in HCC surrounding tissues (P<0.05, γ=0.34). TGF-α was usually existed with HBsAg in regenerated and/or dysplastic liver cells. In the five normal liver tissues, TGF-α and HBsAg were not detectable in hepatocytes and bile ducts.CONCLUSION: Hepatitis B virus infection is closely related with hepatocarcinogenesis. The overexpression of TGF-α in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg. The continued expression of TGF-α might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-α might play a role in morphogenesis and regeneration of intrahepatic bile ducts.

  3. Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

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    Fu Guodong

    2009-12-01

    Full Text Available Abstract Background Transforming growth factor-beta (TGF-beta is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression. Methods To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII and type I receptor (ALK5 receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment. Results and Discussion TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the

  4. Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

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    Davis Rodney

    2006-07-01

    Full Text Available Abstract Background Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF, was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. Methods We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. Results We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM or nucleolin (on the cell surfaces eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of

  5. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer.

    NARCIS (Netherlands)

    Heskamp, S.; Laverman, P.; Rosik, D.; Boschetti, F.; Graaf, W.T.A. van der; Oyen, W.J.G.; Laarhoven, H.W.M. van; Tolmachev, V.; Boerman, O.C.

    2012-01-01

    Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2)(:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors,

  6. MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

    Science.gov (United States)

    Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. BrombergCenter fo...

  7. Hypoxia, leptin, and vascular endothelial growth factor stimulate vascular endothelial cell differentiation of human adipose tissue-derived stem cells.

    Science.gov (United States)

    Bekhite, Mohamed M; Finkensieper, Andreas; Rebhan, Jennifer; Huse, Stephanie; Schultze-Mosgau, Stefan; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2014-02-15

    The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

  8. L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

    Science.gov (United States)

    Trimmel, K; Cvikl, B; Müller, H-D; Nürnberger, S; Gruber, R; Moritz, A; Agis, H

    2015-03-01

    To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P  0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  9. Implication of STAT3 signaling in human colonic cancer cells during intestinal trefoil factor 3 (TFF3) -- and vascular endothelial growth factor-mediated cellular invasion and tumor growth.

    Science.gov (United States)

    Rivat, Christine; Christine, Rivat; Rodrigues, Sylvie; Sylvie, Rodrigues; Bruyneel, Erik; Erik, Bruyneel; Piétu, Geneviève; Geneviève, Piétu; Robert, Amélie; Amélie, Robert; Redeuilh, Gérard; Gérard, Redeuilh; Bracke, Marc; Marc, Bracke; Gespach, Christian; Christian, Gespach; Attoub, Samir; Samir, Attoub

    2005-01-01

    Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.

  10. Epidemiologic Study of Human Epidermal Growth Factor Receptor 2 Expression in Advanced/Metastatic Gastric Cancer: an Assessment of Human Epidermal Growth Factor Receptor 2 Status in Tumor Tissue Samples of Gastric and Gastro-Esophageal Junction Cancer

    Science.gov (United States)

    Seo, Kyung Won; Jeon, Taeyong; Kim, Sewon; Kim, Sung Soo; Kim, Kwanghee; Suh, Byoung-Jo; Hwang, Sunhwi; Choi, SeongHee; Ryu, Seungwan; Min, Jae Seok; Lee, Young-Joon; Jee, Ye Seob; Chae, Hyeondong

    2017-01-01

    Purpose The Trastuzumab for gastric cancer (GC) trial identified human epidermal growth factor receptor 2 (HER2) as a predictor of successful treatment with trastuzumab (HER2 receptor targeting agent) among patients with advanced/metastatic GC. To date, the prevalence of HER2 overexpression in the Korean population is unknown. The present study aimed to assess the incidence of HER2 positivity among GC and gastroesophageal (GE) junction cancer samples and the relationship between HER2 overexpression and clinicopathological characteristics in Korean patients. Materials and Methods Tumor samples collected from 1,695 patients with histologically proven GC or GE junction enrolled at 14 different hospitals in Korea were examined. After gathering clinicopathological data of all patients, HER2 status was assessed by immunohistochemistry (IHC) at each hospital, and IHC 2+ cases were subjected to silver-enhanced in situ hybridization at 3 central laboratories. Results A total of 182 specimens tested positive for HER2, whereas 1,505 tested negative. Therefore, the overall HER2-positive rate in this study was 10.8% (95% confidence interval=9.3%–12.3%). The HER2-positive rate was higher among intestinal-type cases (17.6%) than among other types, and was higher among patients older than 70 years and 50 years of age, compared to other age groups. Conclusions Our evaluation of the HER2 positivity rate (10.8%) among Korean patients with GC and GE junction indicated the necessity of epidemiological data when conducting studies related to HER2 expression in GC and GE junction.

  11. Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats.

    Science.gov (United States)

    Iznaga Escobar, N; Morales, A M; Ducongé, J; Torres, I C; Fernández, E; Gómez, J A

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t 1/2alpha) of 0.250 h and a mean elimination (t 1/2beta) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99mTc-labeled humanized MAb R3 conjugate in patients should be supported.

  12. Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.

    Science.gov (United States)

    Jones, M; Tussey, L; Athanasou, N; Jackson, D G

    2000-03-17

    The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.

  13. Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells

    Science.gov (United States)

    Trau, Heidi A.; Brännström, Mats; Curry, Thomas E.; Duffy, Diane M.

    2016-01-01

    STUDY QUESTION Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation? SUMMARY ANSWER PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis. WHAT IS KNOWN ALREADY Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis. STUDY DESIGN, SIZE, DURATION Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30–45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18–31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs). PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix. MAIN RESULTS AND THE ROLE OF CHANCE Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18–34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but

  14. Expression of growth differentiation factor 6 in the human developing fetal spine retreats from vertebral ossifying regions and is restricted to cartilaginous tissues.

    Science.gov (United States)

    Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Gulati, Twishi; Fang, Zhimin; Pathmanandavel, Sarennya; Diwan, Ashish D

    2016-02-01

    During embryogenesis vertebral segmentation is initiated by sclerotomal cell migration and condensation around the notochord, forming anlagen of vertebral bodies and intervertebral discs. The factors that govern the segmentation are not clear. Previous research demonstrated that mutations in growth differentiation factor 6 resulted in congenital vertebral fusion, suggesting this factor plays a role in development of vertebral column. In this study, we detected expression and localization of growth differentiation factor 6 in human fetal spinal column, especially in the period of early ossification of vertebrae and the developing intervertebral discs. The extracellular matrix proteins were also examined. Results showed that high levels of growth differentiation factor 6 were expressed in the nucleus pulposus of intervertebral discs and the hypertrophic chondrocytes adjacent to the ossification centre in vertebral bodies, where strong expression of proteoglycan and collagens was also detected. As fetal age increased, the expression of growth differentiation factor 6 was decreased correspondingly with the progress of ossification in vertebral bodies and restricted to cartilaginous regions. This expression pattern and the genetic link to vertebral fusion suggest that growth differentiation factor 6 may play an important role in suppression of ossification to ensure proper vertebral segmentation during spinal development.

  15. Endometrial glands as a source of nutrients, growth factors and cytokines during the first trimester of human pregnancy: A morphological and immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Jauniaux Eric

    2004-07-01

    Full Text Available Abstract Background The maternal circulation to the human placenta is not fully established until 10–12 weeks of pregnancy. During the first trimester the intervillous space is filled by a clear fluid, in part derived from secretions from the endometrial glands via openings in the basal plate. The aim was to determine the activity of the glands throughout the first trimester, and to identify components of the secretions. Methods Samples of human decidua basalis from 5–14 weeks gestational age were examined by transmission electron microscopy and immunohistochemically. An archival collection of placenta-in-situ samples was also reviewed. Results The thickness of the endometrium beneath the implantation site reduced from approximately 5 mm at 6 weeks to 1 mm at 14 weeks of gestation. The glandular epithelium also transformed from tall columnar cells, packed with secretory organelles, to a low cuboidal layer over this period. The lumens of the glands were always filled with precipitated secretions, and communications with the intervillous space could be traced until at least 10 weeks. The glandular epithelium reacted strongly for leukaemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, transforming growth factor beta, alpha tocopherol transfer protein, MUC-1 and glycodelin, and weakly for lactoferrin. As gestation advanced uterine natural killer cells became closely approximated to the basal surface of the epithelium. These cells were also immunopositive for epidermal growth factor. Conclusions Morphologically the endometrial glands are best developed and most active during early human pregnancy. The glands gradually regress over the first trimester, but still communicate with the intervillous space until at least 10 weeks. Hence, they could provide an important source of nutrients, growth factors and cytokines for the feto-placental unit. The endometrium may therefore play a greater role in regulating placental

  16. Treatment with recombinant human insulin-like growth factor-I improves growth in patients with PAPP-A2 deficiency

    DEFF Research Database (Denmark)

    Teresa Muñoz-Calvo, María; Barrios, Vicente; Pozo, Jesús;

    2016-01-01

    CONTEXT: Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that specifically cleaves IGFBP-3 and IGFBP-5. Mutations in the PAPP-A2 gene have recently been shown to cause postnatal growth failure in humans, with specific skeletal features, due to the resulting decrease in IGF...

  17. Evaluation of human epidermal growth factor receptor 2 (HER2) single nucleotide polymorphisms (SNPs) in normal and breast tumor tissues and their link with breast cancer prognostic factors.

    Science.gov (United States)

    Furrer, Daniela; Lemieux, Julie; Côté, Marc-André; Provencher, Louise; Laflamme, Christian; Barabé, Frédéric; Jacob, Simon; Michaud, Annick; Diorio, Caroline

    2016-12-01

    Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with worse prognosis and decreased overall survival in breast cancer patients. The HER2 gene contains several polymorphisms; two of the best-characterized HER2 polymorphisms are Ile655Val and Ala1170Pro. The aim of this study was to evaluate the association between these two HER2 polymorphisms in normal breast and breast cancer tissues and known breast cancer prognostic factors in a retrospective cohort study of 73 women with non-metastatic HER2-positive breast cancer. HER2 polymorphisms were assessed in breast cancer tissue and normal breast tissue using TaqMan assay. Ala1170Pro polymorphism in normal breast tissue was associated with age at diagnosis (p = 0.007), tumor size (p = 0.004) and lymphovascular invasion (p = 0.06). Similar significant associations in cancer tissues were observed. No association between the Ile655Val polymorphism and prognostic factors were observed. However, we found significant differences in the distribution of Ile655Val (p = 0.03) and Ala1170Pro (p = 0.01) genotypes between normal breast and breast tumor tissues. This study demonstrates that only the Ala1170Pro polymorphism is associated with prognostic factors in HER2-positive breast cancer patients. Moreover, our results suggest that both HER2 polymorphisms could play a significant role in carcinogenesis in non-metastatic HER2-positive breast cancer women.

  18. Docetaxel Influences Autocrine of Transforming Growth Factors and Induces Apoptosis in Human Ovarian Cancer Cell Line AO

    Institute of Scientific and Technical Information of China (English)

    Yan Zhang; Ya-li Hu; Yun-ying Cheng

    2006-01-01

    @@ Ovarian cancer is the second most common malignancy of female reproductive tract. Docetaxel shows good clinical efficacy against ovarian cancer.This present study was to investigate the role of docetaxel on apoptosis of ovarian cancer epithelial cell line AO as well as the secretion of transforming growth factor (TGF)-α and TGF-β1 during apoptosis.

  19. Determination of free insulin-like growth factor-I in human serum: comparison of ultrafiltration and direct immunoradiometric assay

    DEFF Research Database (Denmark)

    Frystyk, J; Ivarsen, P; Støving, R K;

    2001-01-01

    Two fundamentally different methods are currently used for the determination of free insulin-like growth factor-I (IGF-I): ultrafiltration by centrifugation (UF) and direct immunoradiometric assay (IRMA). The aim was to evaluate a commercial IRMA (DSL, Webster, TX, USA) and to compare it with UF....

  20. Human epidermal growth factor receptor-2 overexpression and amplification in metastatic and recurrent high grade or type 2 endometrial carcinomas

    Directory of Open Access Journals (Sweden)

    Kato R

    2013-08-01

    Full Text Available Rina Kato,1 Kiyoshi Hasegawa,1 Risa Ishii,1 Akiko Owaki,1 Yutaka Torii,1 Shuko Oe,1 Hiroshi Hirasawa,2 Yoichi Kobayashi,3 Yasuhiro Udagawa1 1Department of Obstetrics and Gynecology, Fujita Health University School of Medicine, Toyoake, Japan; 2Department of Pathology, Fujita Health University School of Medicine, Toyoake, Japan; 3Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Mitaka, Japan Introduction: Human epidermal growth factor receptor (HER-2 overexpression or gene amplification is more common in high-grade or type 2 endometrial carcinomas. We assessed the discordance of HER-2 expression between primary and metastatic or recurrent endometrial carcinomas. Materials and methods: Thirty-six primary, along with 14 metastatic and five recurrent tumors (matched to primaries, pathologically confirmed as high-grade or type 2 endometrial carcinomas, were submitted for immunohistochemistry (IHC for HER-2. Fluorescence in situ hybridization was performed when the tumors showed HER-2 overexpression (≥2+ IHC score. The results of the IHC and fluorescence in situ hybridization assays were compared between the primary and metastatic or recurrent tumors. The relationships between HER-2 expression and clinicopathological factors or prognosis were investigated. Results: HER-2 overexpression and HER-2 amplification (a ratio of HER-2 copies to chromosome 17 [CEP17] copies ≥2.2 were detected in 33.3% (twelve of 36 patients and 5.6% (two of 36 patients of primary tumors, respectively. HER-2 overexpression was not associated with clinicopathological factors or prognosis. In 19 tumor specimens obtained from metastatic or recurrent tumors, HER-2 overexpression and HER-2 amplification were detected in 57.9% (eleven patients and 15.8% (three patients, respectively. HER-2 overexpression tended to predict a worse prognosis. Conclusion: HER-2 expression in metastatic or recurrent tumors was more frequent than in matched primary high

  1. Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

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    Surena Vahabi

    2016-01-01

    Full Text Available Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF and platelet-rich fibrin (PRF on proliferation and viability of human gingival fibroblasts (HGFs.Materials and Methods: Anitua's PRGF and Choukran's PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-values<0.05 were considered statistically significant.Results: PRGF treatment induced statistically significant (P<0.001 proliferation of HGF cells compared to the negative control (100% viability at 24, 48 and 72 hours in values of 123%±2.25%, 102%±2.8% and 101%±3.92%, respectively. The PRF membrane treatment of HGF cells had a statistically significant effect on cell proliferation (21%±1.73%, P<0.001 at 24 hours compared to the negative control. However, at 48 and 72 hours after treatment, PRF had a negative effect on HGF cell proliferation and caused 38% and 60% decrease in viability and proliferation compared to the negative control, respectively. The HGF cell proliferation was significantly higher in PRGF than in PRF group (P< 0.001.Conclusion: This study demonstrated that PRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF.

  2. Fibroblast Growth Factor 2 Regulates High Mobility Group A2 Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Kalomoiris, Stefanos; Cicchetto, Andrew C; Lakatos, Kinga; Nolta, Jan A; Fierro, Fernando A

    2016-09-01

    Mesenchymal stem cells (MSCs) are an excellent source for numerous cellular therapies due to their simple isolation, low immunogenicity, multipotent differentiation potential and regenerative secretion profile. However, over-expanded MSCs show decreased therapeutic efficacy. This shortcoming may be circumvented by identifying methods that promote self-renewal of MSCs in culture. HMGA2 is a DNA-binding protein that regulates self-renewal in multiple types of stem cells through chromatin remodeling, but its impact on human bone marrow-derived MSCs is not known. Using an isolation method to obtain pure MSCs within 9 days in culture, we show that expression of HMGA2 quickly decreases during early expansion of MSCs, while let-7 microRNAs (which repress HMGA2) are simultaneously increased. Remarkably, we demonstrate that FGF-2, a growth factor commonly used to promote self-renewal in MSCs, rapidly induces HMGA2 expression in a time- and concentration-dependent manner. The signaling pathway involves FGF-2 receptor 1 (FGFR1) and ERK1/2, but acts independent from let-7. By silencing HMGA2 using shRNAs, we demonstrate that HMGA2 is necessary for MSC proliferation. However, we also show that over-expression of HMGA2 does not increase cell proliferation, but rather abrogates the mitogenic effect of FGF-2, possibly through inhibition of FGFR1. In addition, using different methods to assess in vitro differentiation, we show that modulation of HMGA2 inhibits adipogenesis, but does not affect osteogenesis of MSCs. Altogether, our results show that HMGA2 expression is associated with highly proliferating MSCs, is tightly regulated by FGF-2, and is involved in both proliferation and adipogenesis of MSCs. J. Cell. Biochem. 117: 2128-2137, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21 modified with polyethylene glycol.

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    Zhifeng Huang

    Full Text Available As one of fibroblast growth factor (FGF family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21 was modified with polyethylene glycol (PEGylation in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients.

  4. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. (Genentech, Inc., South San Francisco, CA (USA))

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  5. Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Xue; Kumru, Ozan S.; Blaber, Sachiko I.; Middaugh, C. Russell; Li, Ling; Ornitz, David M.; Sutherland, Mason A.; Tenorio, Connie A.; Blaber, Michael (FSU); (WU-MED); (Kansas)

    2016-07-06

    Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

  6. Pharmacokinetics of recombinant human basic fibroblast growth factor in rabbits and mice serum and rabbits aqueous humor

    Institute of Scientific and Technical Information of China (English)

    Qi ZHANG; Guang-ji WANG; Jian-guo SUN

    2004-01-01

    AIM: To study the pharmacokinetics of recombinant human basic fibroblast growth factor (rhbFGF) in rabbits and mice after iv and postocular administration, and the changes of rhbFGF in rabbits aqueous humor after postocular administration. METHODS: After iv or postocular administration three doses of rhbFGF in rabbits and mice,rhbFGF concentration in serum and rabbit aqueous humor was determined by enzyme-linked immunosorbent .assay. RESULTS: Serum concentration-time data of rabbits after iv administration of rhbFGF 1, 2, and 4 μg/kg were fitted to bi-exponential equations with half-lives of 0.9, 0.9, and 0.6 min for T1/2α and 7, 8, and 4.7 min for T1/2β.Plasma concentration-time data of mice after iv administration of rhbFGF 2.5, 5 and 10 μg/kg were fitted to biexponential equations with half-lives of 0.4, 0.6, and 0.9 min for T1/2α and 6, 5, and 7 min for T1/2β. The AUCs were linearly correlated to doses in both cases (rrabbit=0.997, rmouse=0.999). The serum concentrations of rhbFGF were very low, near to the background after postocular administration of 2 or 5 μg/kg, in both rabbits and mice. The rhbFGF levels in rabbits aqueous humor were higher than control 8 h postdose (P<0.01). CONCLUSION: rhbFGF within the examined doses had a linear pharmacokinetics in rabbits and mice. High concentration of rhbFGF was found in rabbits aqueous humor after postocular administration.

  7. Downregulation of Epidermal Growth Factor Receptor Expression Contributes to α-TEA's Proapoptotic Effects in Human Ovarian Cancer Cell Lines

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    Ming-Chieh Shun

    2010-01-01

    Full Text Available RRR-α-tocopherol derivative α-TEA (RRR-α-tocopherol ether-linked acetic acid analog has been shown to be a potent antitumor agent both in vivo and in vitro. In this study, we investigated the effects of α-TEA on the expression of epidermal growth factor receptor (EGFR family members, ErbB1, 2 and 3, and the role of ErbB 2 and 3 in α-TEA-induced apoptosis and suppression of Akt, FLIP and survivin in the cisplatin-sensitive (A2780S and -resistant (A2780/CP70R human ovarian cancer cell lines. Data show that α-TEA's ability to induced apoptosis was associated with reduced expression of ErbB1 (cisplatin-resistant cells, 2 and 3 (both cell types and reduced levels of the phosphorylated (active form of Akt; as well as, reduced levels of FLIP and survivin proteins in both cell types. Ectopic overexpression and siRNA knockdown studies showed that ErbB2, ErbB3, Akt, FLIP and survivin are involved in α-TEA-induce apoptosis and that α-TEA downregulates FLIP and survivin via suppression of pAkt, which is mediated by ErbB2 and ErB3. Thus, α-TEA is a potent pro-apoptotic agent for both cisplatin-sensitive and -resistant ovarian cancer cell lines in cell culture and it produces cell death, at least in part, by downregulation of members of the EGFR family.

  8. Fibroblast growth factor-20 increases the yield of midbrain dopaminergic neurons derived from human embryonic stem cells

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    Ana Sofia Correia

    2007-12-01

    Full Text Available In the central nervous system, fibroblast growth factor (FGF-20 has been reported to act preferentially on midbrain dopaminergic neurons. It also promotes the dopaminergic differentiation of stem cells. We have analyzed the effects of FGF-20 on human embryonic stem cells (hESCs differentiation into dopaminergic neurons. We induced neuronal differentiation of hESCs by co-culturing those with PA6 mouse stromal cells for 3 weeks. When we supplemented the culture medium with FGF-20, the number of tyrosine hydroxylase (TH- expressing neurons increased fivefold, from 3% to 15% of the hESC-derived cells. The cultured cells also expressed other midbrain dopaminergic markers (PITX3, En1, Msx1, and Aldh1, suggesting that some had differentiated into midbrain dopaminergic neurons. We observed no effect of FGF-20 on the size of the soma area or neurite length of the TH-immunopositive neurons. Regardless of whether FGF-20 had been added or not, 17% of the hESC-derived cells expressed the pan-neuronal marker b-III-Tubulin. The proportion of proliferating cells positive for Ki-67 was also not affected by FGF-20 (7% of the hESC-derived cells. By contrast, after 3 weeks in culture FGF-20 significantly reduced the proportion of cells undergoing cell death, as revealed by immunoreactivity for cleaved caspase-8, Bcl-2 associated X protein (BAX and cleaved caspase-3 (2.5% to 1.2% of cleaved caspase-3-positive cells out of the hESC-derived cells. Taken together, our results indicate that FGF-20 specifically increases the yield of dopaminergic neurons from hESCs grown on PA6 feeder cells and at least part of this effect is due to a reduction in cell death.

  9. Pertuzumab for the treatment of patients with human epidermal growth factor receptor 2-positive breast cancer in Japan.

    Science.gov (United States)

    Osako, Tomofumi; Nishimura, Reiki; Nishiyama, Yasuyuki; Fujisue, Mamiko

    2015-11-01

    Pertuzumab, a novel anti-human epidermal growth factor receptor 2 (HER2) agent, is effective for metastatic HER2-positive breast cancer when used in combination with taxane and trastuzumab. The aim of the present study was to describe the use of pertuzumab in Japan. A phase I clinical trial of pertuzumab for HER2-positive metastatic breast cancer was first conducted in the United States in 2001 (study ID no. TOC2297g) and for HER2-positive solid cancers in Japan in 2004 (study ID no. JO17076). However, Japanese patients were not enrolled in a global phase II trial for metastatic breast cancer (study ID no. BO17929) and no phase II trial of pertuzumab for Japanese patients has yet been conducted. A phase III trial on pertuzumab for metastatic breast cancer (CLEOPATRA study), which included 53 Japanese patients, revealed that pertuzumab significantly prolonged progression-free and overall survival. However, the superiority of the pertuzumab group was not verified in the subgroup analysis of Japanese patients, which was not a preplanned analysis. Therefore, a postmarketing clinical trial for Japanese patients with HER2-positive metastatic breast cancer (COMACHI study) was initiated in November, 2013, to investigate the clinical effectiveness of pertuzumab in Japanese patients. As of December, 2014, global trials on pertuzumab in the metastatic and adjuvant settings are currently ongoing. These trials included Japanese patients with HER2-positive breast cancer. Pertuzumab was approved in Japan in August, 2013 due to the positive findings of the CLEOPATRA study. Unlike the United States and Europe, the Japanes Pharmaceutical and Medical Devices Agency approved the administration of pertuzumab as second- or later-line treatment for HER2-positive metastatic breast cancer, as well as first-line treatment. Furthermore, pertuzumab may be used in combination with other chemotherapeutic agents, with the exception of docetaxel. The approval of the expanded use of pertuzumab is

  10. Kidney growth in normal and diabetic mice is not affected by human insulin-like growth factor binding protein-1 administration

    NARCIS (Netherlands)

    V. Cingel-Ristic; B.F. Schrijvers; A.K. van Vliet (Arlène); R. Rasch; V.K. Han; S.L.S. Drop (Stenvert); A. Flyvbjerg (Allan)

    2005-01-01

    textabstractInsulin-like growth factor I (IGF-I) accumulates in the kidney following the onset of diabetes, initiating diabetic renal hypertrophy. Increased renal IGF-I protein content, which is not reflected in messenger RNA (mRNA) levels, suggests that renal IGF-I accumulation is

  11. Effect of hepatocyte growth factor and transforming growth factor-β1 on atrial fibroblasts fibrosis

    Institute of Scientific and Technical Information of China (English)

    张建成

    2012-01-01

    Objective To investigate the effect of hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGFβ1) on the expression of α-smooth muscle actin(α-SMA) and collagen I in human atrial fibroblast in vitro, and to explore the possible molecular mechanism of atrial fibrosis in patients

  12. Effects of Nerve Growth Factor on Proliferation and DNA Synthesis of Cultured Human Fetal Retinal Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Wensheng Li; Jun Wen; Deyong Jiang; Jianguang Ding

    2002-01-01

    Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.

  13. Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells.

    Science.gov (United States)

    Dvorak, Petr; Dvorakova, Dana; Koskova, Stanislava; Vodinska, Martina; Najvirtova, Miroslava; Krekac, Daniel; Hampl, Ales

    2005-09-01

    Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this

  14. Preliminary separation of the growth factors in platelet-rich plasma: effects on the proliferation of human marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    HUANG Qian; WANG Yun-dan; WU Tao; JIANG Shan; HU Yan-ling; PEI Guo-xian

    2009-01-01

    Background Platelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma. Methods The gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor 131 (TGF-β1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results PRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-β1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P 0.05). Fraction A and D showed no significant difference to the negative control group (P >0.05). Conclusions The growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.

  15. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.

    Science.gov (United States)

    Ogitani, Yusuke; Hagihara, Katsunobu; Oitate, Masataka; Naito, Hiroyuki; Agatsuma, Toshinori

    2016-07-01

    Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer

  16. Effect of vascular endothelial growth factor and its receptor KDR on human airway smooth muscle cells proliferation

    Institute of Scientific and Technical Information of China (English)

    ZOU hui; XU Yong-jian; ZHANG Zhen-xiang

    2005-01-01

    @@ Airway remodeling with inflammatory cell infiltration, epithelial shedding, basement membrane thickening and increased mass of airway smooth muscle (ASM) is an important determinant of bronchial obstruction and hyperresponsiveness in asthma.1,2 Increased ASM mass is by far the most important abnormality responsible for excessive airway narrowing and compliance of the airway wall in asthma.1-3 ASM growth and proliferation in asthma is a complex phenomenon of which the underlying mechanisms are difficult to investigate in vivo. The increased amount of ASM in asthmatics is an indication of abnormal cell proliferation and growth, but little is known regarding the molecular mechanisms and factors that regulate ASM cell proliferation and growth in asthma.

  17. Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells.

    Science.gov (United States)

    Li, Fang; Cui, Jinquan

    2015-07-01

    Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression.

  18. The response to epidermal growth factor of human maxillary tumor cells in terms of tumor growth, invasion and expression of proteinase inhibitors.

    Science.gov (United States)

    Mizoguchi, H; Komiyama, S; Matsui, K; Hamanaka, R; Ono, M; Kiue, A; Kobayashi, M; Shimizu, N; Welgus, H G; Kuwano, M

    1991-11-11

    Three cancer cell lines, IMC-2, IMC-3 and IMC-4, were established from a single tumor of a patient with maxillary cancer. We examined responses to epidermal growth factor (EGF) of these 3 cell lines with regard to cell growth and tumor invasion. The growth rate of IMC-2 in nude mice was markedly faster than that of the IMC-3 and IMC-4 cell lines. Assay for invasion through fibrin gels showed significantly enhanced invasive capacity of IMC-2 cells in response to EGF, but no change for IMC-3 and IMC-4 cells. We examined response to EGF of IMC-2 cells with regard to expression of a growth-related oncogene (c-fos), proteinases and their inhibitors. Expression of c-fos was transiently increased in IMC-2 cells at rates comparable to those seen in the 2 other lines in the presence of EGF. There was no apparent effect of EGF on the expression of urokinase-type plasminogen activator and 72-kDa type-IV collagenase in IMC-2 cells. In contrast, EGF specifically enhanced the expression of plasminogen activator inhibitor-I (PAI-I) and tissue inhibitor of metalloproteinases-I (TIMP-I) in IMC-2 cells. Our data suggest that proteinase inhibitors or other related factors may play an important role in tumor growth and invasion in response to EGF.

  19. Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation.

    Science.gov (United States)

    Lassarre, C; Hardouin, S; Daffos, F; Forestier, F; Frankenne, F; Binoux, M

    1991-03-01

    IGF-I, IGF-II, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38- to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-II levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term. Correlations were found between fetal placental lactogen levels and those of IGF-I and IGF-II, which is consistent with the hypothesis that placental lactogen is involved in the regulation of IGF synthesis in the fetus. With weight (either measured at birth or deduced from echographical data) as index of fetal size, IGF-I levels were significantly (p less than 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF-II levels were similar in the two groups. Similarly, IGF-I (but not IGF-II) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age (p less than 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF-II) is involved in the control of fetal size. Total fetal BP concentrations were approximately 1/3 those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Growth hormone secretion from chicken adenohypophyseal cells in primary culture: effects of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release.

    Science.gov (United States)

    Perez, F M; Malamed, S; Scanes, C G

    1987-03-01

    A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release; however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect, increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical.

  1. The Expression of Sprouty1, an Inhibitor of Fibroblast Growth Factor Signal Transduction, Is Decreased in Human Prostate Cancer

    Science.gov (United States)

    2004-07-15

    contribute to prostate cancer pro- gression. Yan et al. (4) have shown in the Dunning rat model system that as these transplantable tumors progress...mediated by the FGFR and the epidermal growth factor receptor during eye development and oogenesis in Drosophila (17–19). During Drosophila eye development...treated with bovine antigoat IgG (1:5000; Santa Cruz Biotechnology) or rat antimouse IgG secondary antibody conjugated to horseradish peroxidase (1

  2. Iloprost up-regulates vascular endothelial growth factor expression in human dental pulp cells in vitro and enhances pulpal blood flow in vivo.

    Science.gov (United States)

    Limjeerajarus, Chalida Nakalekha; Osathanon, Thanaphum; Manokawinchoke, Jeeranan; Pavasant, Prasit

    2014-07-01

    Prostacyclin (PGI2) is a biomolecule capable of enhancing angiogenesis and cellular proliferation. We investigated the influence of a PGI2 analogue (iloprost) on dental pulp revascularization in vitro and in vivo by using human dental pulp cells (HDPCs) and a rat tooth injury model, respectively. Iloprost stimulated the human dental pulp cell mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF) in a significant dose-dependent manner. This mRNA up-regulation was significantly inhibited by pretreatment with a PGI2 receptor antagonist and forskolin (a protein kinase A activator). In contrast, a protein kinase A inhibitor significantly enhanced the iloprost-induced mRNA expression of VEGF, FGF-2, and PDGF. Pretreatment with a fibroblast growth factor receptor inhibitor attenuated the VEGF, FGF-2, and PDGF mRNA expression, indicating opposing regulatory mechanisms. The effect of iloprost on the dental pulp was investigated in vivo by using a rat molar pulp injury model. The iloprost-treated group exhibited a significant increase in pulpal blood flow at 72 hours compared with control. The present study indicates that iloprost may be a candidate agent to promote neovascularization in dental pulp tissue, suggesting the potential clinical use of iloprost in vital pulp therapy. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. Phase III randomized study comparing docetaxel plus trastuzumab with vinorelbine plus trastuzumab as first-line therapy of metastatic or locally advanced human epidermal growth factor receptor 2-positive breast cancer: the HERNATA study

    DEFF Research Database (Denmark)

    Andersson, Michael; Lidbrink, Elisabeth; Bjerre, Karsten;

    2011-01-01

    To evaluate docetaxel or vinorelbine, both with trastuzumab, as first-line therapy of human epidermal growth factor receptor 2-positive advanced breast cancer.......To evaluate docetaxel or vinorelbine, both with trastuzumab, as first-line therapy of human epidermal growth factor receptor 2-positive advanced breast cancer....

  4. Factors affecting disease-free survival in patients with human epidermal growth factor receptor 2-positive breast cancer who receive adjuvant trastuzumab

    Science.gov (United States)

    GÜNDÜZ, SEYDA; GÖKSU, SEMA SEZGIN; ARSLAN, DENIZ; TATLI, ALI MURAT; UYSAL, MÜKREMIN; GÜNDÜZ, UMUT RIZA; SEVINÇ, MERT MAHSUNI; COŞKUN, HASAN SENOL; BOZCUK, HAKAN; MUTLU, HASAN; SAVAS, BURHAN

    2015-01-01

    Breast cancer is the most frequently diagnosed cancer in women worldwide and the second cause of cancer-related mortality. A total of 20–30% of patients with early-stage breast cancer develop recurrence within the first 5 years following diagnosis. Trastuzumab significantly improves overall survival and disease-free survival (DFS) in women with human epidermal growth factor receptor 2 (HER2)-positive early and locally advanced breast cancer. This study aimed to determine the factors that affect DFS following adjuvant transtuzumab therapy. A total of 62 patients treated with trastuzumab for early and locally advanced breast cancer were included in our study. Data, including pathology, treatment and treatment outcome, rate of recurrence and laboratory tests, were retrospectively collected. There was no significant association between DFS and age, menopausal status, disease stage and hormone receptor status. The median follow-up was 48.4 months. The median DFS of patients treated with adjuvant trastuzumab was 64.1 months. In addition, the median DFS was 44.3 vs. 66.8 months in patients with platelet-lymphocyte ratio (PLR) ≤200 vs. >200, respectively (log-rank test; P=0.001), and 70 vs. 45 months in patients with eosinophil count ≤70 vs. >70×103/mm3 (log-rank test; P=0.001). Our data revealed the prognostic relevance of a decrease in the peripheral blood eosinophil count and PLR value following trastuzumab therapy in breast cancer. PLR and eosinophil count measurements are cost-effective, readily available worldwide, non-invasive and safe. Combined with other markers, such as patient age, tumor stage and tumor histology, may be effectively used for patients with breast cancer. PMID:26623060

  5. Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 徐永健; 张珍祥; 许淑云; 倪望; 陈士新

    2004-01-01

    Summary: In order to investigate the effect of nuclear factor-kappa B (NF-κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. The NF-κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBa protein expression was measured by Western blot.RT-PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF-κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF-κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups (P<0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups (P<0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration-dependent manner in hypoxia. In conclusion, NF-κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF-κB activation can decrease the VEGF mRNA expression. h is suggested that the activation of NF-κB is involved in the VEGFmRNA expression of HPASMCs under hypoxia.

  6. Novel angiogenic inhibitor DN-9693 that inhibits post-transcriptional induction of connective tissue growth factor (CTGF/CCN2) by vascular endothelial growth factor in human endothelial cells.

    Science.gov (United States)

    Kondo, Seiji; Tanaka, Noriko; Kubota, Satoshi; Mukudai, Yoshiki; Yosimichi, Gen; Sugahara, Toshio; Takigawa, Masaharu

    2006-01-01

    Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)-mediated induction of the ctgf/ccn2 gene was a post-transcriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.

  7. Pertuzumab for the treatment of patients with human epidermal growth factor receptor 2-positive breast cancer in Japan

    Science.gov (United States)

    OSAKO, TOMOFUMI; NISHIMURA, REIKI; NISHIYAMA, YASUYUKI; FUJISUE, MAMIKO

    2015-01-01

    Pertuzumab, a novel anti-human epidermal growth factor receptor 2 (HER2) agent, is effective for metastatic HER2-positive breast cancer when used in combination with taxane and trastuzumab. The aim of the present study was to describe the use of pertuzumab in Japan. A phase I clinical trial of pertuzumab for HER2-positive metastatic breast cancer was first conducted in the United States in 2001 (study ID no. TOC2297g) and for HER2-positive solid cancers in Japan in 2004 (study ID no. JO17076). However, Japanese patients were not enrolled in a global phase II trial for metastatic breast cancer (study ID no. BO17929) and no phase II trial of pertuzumab for Japanese patients has yet been conducted. A phase III trial on pertuzumab for metastatic breast cancer (CLEOPATRA study), which included 53 Japanese patients, revealed that pertuzumab significantly prolonged progression-free and overall survival. However, the superiority of the pertuzumab group was not verified in the subgroup analysis of Japanese patients, which was not a preplanned analysis. Therefore, a postmarketing clinical trial for Japanese patients with HER2-positive metastatic breast cancer (COMACHI study) was initiated in November, 2013, to investigate the clinical effectiveness of pertuzumab in Japanese patients. As of December, 2014, global trials on pertuzumab in the metastatic and adjuvant settings are currently ongoing. These trials included Japanese patients with HER2-positive breast cancer. Pertuzumab was approved in Japan in August, 2013 due to the positive findings of the CLEOPATRA study. Unlike the United States and Europe, the Japanes Pharmaceutical and Medical Devices Agency approved the administration of pertuzumab as second- or later-line treatment for HER2-positive metastatic breast cancer, as well as first-line treatment. Furthermore, pertuzumab may be used in combination with other chemotherapeutic agents, with the exception of docetaxel. The approval of the expanded use of pertuzumab is

  8. Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab's efficacy in vitro?

    Directory of Open Access Journals (Sweden)

    Hurrell T

    2013-06-01

    Full Text Available Tracey Hurrell, Kim OuthoffDepartment of Pharmacology, University of Pretoria, Pretoria, South AfricaIntroduction: Despite trastuzumab having enhanced selectivity for human epidermal growth factor receptor 2 (HER-2 overexpressing breast cancer cells, treatment is hampered by interindividual variation and tumors with high mitogenic potential. The lack of significant clinical benefit in certain patient cohorts suggests that HER-2 expression is ineffective as a sole prognostic indicator of response to therapy. Therefore, optimizing the clinical role of trastuzumab in drug combinations remains critical for clinical success.Aim: To investigate the effects of trastuzumab in combination with either doxorubicin or geldanamycin on in vitro cell viability, cell cycling, apoptosis and relative HER-2 expression in HER-2-positive (SK-BR-3 and estrogen receptor-positive (MCF-7 breast adenocarcinoma models.Results: HER-2-rich SK-BR-3 cells demonstrated a greater sensitivity to the effects of doxorubicin than MCF-7 cells. Concurrent trastuzumab exposure resulted in a further reduction in cell viability. This decreased cell viability induced by doxorubicin was associated with activation of executioner caspases as well as with alterations in cell-cycle kinetics, primarily promoting S-phase accumulation. Doxorubicin had no effect on surface HER-2 density expression. Geldanamycin reduced cell viability significantly greater in SK-BR-3 than MCF-7 cells, and was associated with G2 cell-cycle accumulation. The addition of trastuzumab did not augment these effects. Geldanamycin promoted substantial reductions in relative surface HER-2 density in SK-BR-3 cells.Conclusion: The in vitro data supported the rationale for using doxorubicin in trastuzumab-based therapies. Therefore, despite the incidence of cardiotoxicity, doxorubicin could retain a fundamental role in treating HER-2-positive breast cancer. While geldanamycin is a potent cytotoxic agent, its concurrent use

  9. Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, Fumiaki, E-mail: f_naka@nirs.go.jp [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Umeda, Sachiko [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Yasuda, Takeshi [Department of Radiation Emergency Medicine, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba (Japan); Asada, Masahiro; Motomura, Kaori; Suzuki, Masashi [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Zakrzewska, Malgorzata [Faculty of Biotechnology, University of Wroclaw (Poland); Imamura, Toru [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Imai, Takashi [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan)

    2013-02-01

    Purpose: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. Methods and Materials: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with {gamma}-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. Results: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. Conclusion: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal

  10. Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cell transplantation improves hypoxic-ischemic brain injury

    Institute of Scientific and Technical Information of China (English)

    Dengna Zhu; Yanjie Jia; Jun Wang; Boai Zhang; Guohui Niu; Yazhen Fan

    2011-01-01

    Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cells were transplanted into a hypoxic-ischemic neonatal rat model via the tail vein.BrdU-positive cells at day 7post-transplantation,as well as nestin-and neuron specific enolase-positive cells at day 14 wereincreased compared with those of the single neural stem cell transplantation group.In addition,theproportion of neuronal differentiation was enhanced.The genetically modified cell-transplanted ratsexhibited enhanced performance in correctly crossing a Y-maze and climbing an angled slope compared with those of the single neural stem cell transplantation group.These results showed that human insulin-like growth factor 1-transfected neural stem cell transplantation promotes therecovery of the learning,memory and motor functions in hypoxic-ischemic rats.

  11. Growth factors and new periodontology

    Directory of Open Access Journals (Sweden)

    Paknejad M

    1999-06-01

    Full Text Available Growth factors are biological mediators that have a key roll in proliferation, chemotaxy and"ndifferentiation by acting on specific receptors on the surface of cells and regulating events in wound"nhealing.They can be considered hormones that are not released in to the blood stream but have one a"nlocal action. Some of these factors can regulate premature change in GO to Gl phase in cell devesion"ncycle and even may stimulate synthesis of DNA in suitable cells, Growth substances, primarily secreted"nby fibroblasts, endothelia! cells, macrophages and platelet, include platelet derived growth factor"n(PDGF, insulin like growth factor (IGF transforming growth factor (TGFa and (3 and bone"nmorphogenetic proteins BMPs that approximately are the most important of them. (BMPs could be"nused to control events during periodontal, craniofacial and implant wound healing through favoring bone"nformation"nAccording toLynch, combination of PGDF and IGF1 would be effective in promoting growth of all the"ncomponents of the periodontium."nThe aim of this study was to characterize growth factor and review the literature to determine the"nmechanism of their function, classification and application in implant and periodontal treatment.

  12. Effect of CPU-XT-008, a combretastatin A-4 analogue, on the proliferation, apoptosis and expression of vascular endothelial growth factor and basic fibroblast growth factor in human umbilical vein endothelial cells.

    Science.gov (United States)

    Xiong, Rui; Sun, Jing; Liu, Kun; Xu, Yungen; He, Shuying

    2016-01-01

    The present study investigated the effect of the combretastatin A-4 analogue CPU-XT-008 on the proliferation, apoptosis and expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) in human umbilical vein endothelial cells (HUVECs). The proliferation capacity of HUVECs was analyzed with a cell viability assay, while their apoptosis and migration abilities were evaluated via flow cytometry and monolayer denudation assay, respectively. The mRNA and protein expression levels of VEGF and FGF-2 in these cells were determined by reverse transcription-polymerase chain reaction, and cell-based ELISA, western blotting and immunocytochemistry, respectively. The results demonstrated that CPU-XT-008 inhibited proliferation and migration, and induced apoptosis in HUVECs in a dose-dependent manner. In addition, CPU-XT-008 downregulated the mRNA and protein expression levels of VEGF and FGF-2 in these cells. These findings suggest that CPU-XT-008 exerts anti-angiogenic effects in HUVECs, which may explain the inhibition of cell proliferation and migration, induction of apoptosis, and reduction in the mRNA and protein expression levels of VEGF and FGF-2 observed in the present study.

  13. Changes of expression of estrogen and progestrone receptors, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy in the treatment of breast cancer.

    Science.gov (United States)

    Li, M L; Dong, Y; Luan, S L; Zhao, Z H; Ning, F L

    2016-01-01

    Recent studies suggest that the development and prognosis of breast cancer is in close correlation to molecular subtype of breast cancer. Neoadjuvant chemotherapy has been extensively applied in the treatment of local breast cancer in advanced stage. In order to verify the correlation between expression changes of estrogen receptor, progestrone receptor, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy and neoadjuvant chemotherapy, we studied 120 patients with stage IIAIIIC breast cancer who underwent neoadjuvant chemotherapy in Binzhou Medical University Hospital, Shandong, China from February 2011 to February 2015. Clinical characteristics were retrospectively analyzed. The expression of estrogen receptor, progesterone receptor, human epithelial growth factor receptor 2 and Ki-67 of patients were detected using the immunohistochemical method before and after neoadjuvant chemotherapy. The results suggest that the overall remission rate of neoadjuvant chemotherapy was 76.7% (92/120) of which 16.7% (20/120) of cases had complete remission, 60% (72/120) had partial remission and 23.3% (28/120) were stable. There were no cases of progressive disease. The property of estrogen receptor and the expression of Ki-67 of primary tumor were correlated to the remission rate of neoadjuvant chemotherapy (P less than 0.05). The expression of Ki-67 had a significant decline after neoadjuvant chemotherapy, and the difference had statistical significance (P less than 0.05). The difference in expression of estrogen receptor, progesterone receptor and human epithelial growth factor receptor 2 before and after neoadjuvant chemotherapy had statistical significance (P > 0.05). Hence, it can be concluded that breast cancer patients with negative estrogen receptor expression and high Ki-67 expression before neoadjuvant chemotherapy can achieve better curative effects. Neoadjuvant chemotherapy cannot change the expression states of estrogen receptor

  14. Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine

    OpenAIRE

    2015-01-01

    Introduction Periodontitis is one of the most widespread infectious diseases in humans. We previously promoted significant periodontal tissue regeneration in swine models with the transplantation of autologous periodontal ligament stem cells (PDLSCs) and PDLSC sheet. We also promoted periodontal tissue regeneration in a rat model with a local injection of allogeneic bone marrow mesenchymal stem cells. The purpose of the present study is to investigate the roles of the hepatocyte growth factor...

  15. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane.

    Science.gov (United States)

    Russo, Alessandra; Bonci, Paola; Bonci, Paolo

    2012-06-01

    The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

  16. A sensitive enzyme immunoassay for human epidermal growth factor. Determination of hEGF in human serum and urine and pharmacokinetics in mouse.

    Science.gov (United States)

    Hayashi, T; Hashimoto, K; Sakamoto, S

    1989-07-01

    A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.

  17. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N;

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...... cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract...... for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature...

  18. [The expression of transcription factors Snail and Slug in epithelial-mesenchymal transition of human lens epithelial cells induced by transforming growth factor-β2].

    Science.gov (United States)

    Wang, Y N; Pei, C; Qin, L; Li, J M; Yi, J L; Chen, L

    2016-04-11

    To investigate the expression of transcription factors snail and slug in epithelial mesenchymal transition (EMT) of human lens epithelial cells (HLEC) induced by transforming growth factor-β2 (TGF-β2). Experimental research. HLEC were treated with different concentrations of TGF-β2 (1.0 and 10.0 μg/L) for different time. The morphological changes were observed under inverted microscope. The expression and cellular localization of snail and slug were evaluated by immunofluorescence. Expressions of snail, slug, E-Cadherin and α-SMA were further determined by Western blot analysis. Single factor analysis of variance, rank sum test and Pearson correlation were used for statistical analysis. Cultured HLEC were polygonal monolayer cells with tight intercellular adhesion closely and patchy distribution. After treatment of different doses of TGF-β2 for 24 h, HLEC became isolated, exhibited long spindle-like shape as fibroblastic phenotype. The immunofluorescence staining indicated that snail and slug were localized in the nuclei. The expressions of snail and slug appeared to be positive correlative to TGF-β2 dose (snail protein expression: 0.74±0.16, 1.13±0.03, 1.54±0.18 and slug protein expression: 1.96±0.02, 3.12±0.09, 4.07±0.12 in HLEC treated with 0.1, 1.0 and 10 μg/L TGF-β2 respectively) (χ(2)=9.62,P=0.022;F=241.10,Psnail and slug in HLEC were also increased with extending duration of TGF-β2 (1.0 μg/L). The expression levels of both proteins were modestly up-regulated at 8 hours, robustly increased at 24 h, reached peak at 48h and began to decline at 72 h (snail protein expression: 0.90±0.13, 1.43±0.14, 1.96±0.27, 1.57±0.16 and slug protein expression: 0.91±0.36, 1.24±0.16, 2.44±0.26, 1.43±0.16 in HLEC treated with 1.0 μg/L TGF-β2 for 8 h, 24 h, 48 h and 72 h respectively) (F=12.49,P=0.001;F=14.03,Psnail and slug might be time and dose-dependently involved in in-vitro TGF-β2-induced EMT of HLEC. (Chin J Ophthalmol, 2016, 52: 285-290).

  19. The Difference between Growth Factor Expression after Single and Multiple Fractures: Preliminary Results in Human Fracture Healing

    Science.gov (United States)

    Binder, Harald; Eipeldauer, Stefan; Gregori, Markus; Höchtl-Lee, Leonard; Thomas, Anita; Tiefenboeck, Thomas M.; Hajdu, Stefan; Sarahrudi, Kambiz

    2015-01-01

    Objectives. Circulating levels of VEGF-A (Vascular Endothelia Growth Factor-A), TGF-β1 (Transforming Growth Factor-beta 1), and M-CSF (Macrophage-Colony Stimulating Factor) were found to be predictors of bone healing and therefore prognostic criteria of delayed bone healing or nonunion. The aim of this study was to evaluate a potential rise of these markers in patients with multiple fractures of long bones compared to patients with single fractured long bone. Methods. 92 patients were included in the study and finally after excluding all female patients 45 male patients were left for final analysis and divided into the single or multiple fracture group. TGF-β1, M-CSF, and VEGF-A serum levels were analysed over a time period of two weeks. Results. MCSF serum concentrations were higher in the group with multiple fractures as also TGF-β1 serum concentrations were at one and two weeks after trauma. No statistically significant difference was observed in the VEGF-A serum concentrations of both groups at either measurement point. Conclusion. We did observe a correlation between the quantity of the M-CSF and TGF-β1 expressions in serum and the number of fractured bones; surprisingly there was no statistically significant difference in the serum levels between patients with single and multiple fractures of long bones. PMID:26246654

  20. Epidermal Growth Factor Receptor Expression Modulates Antitumor Efficacy of Vandetanib or Cediranib Combined With Radiotherapy in Human Glioblastoma Xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Wachsberger, Phyllis R., E-mail: Phyllis.wachsberger@jeffersonhospital.org [Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania (United States); Lawrence, Yaacov R.; Liu Yi; Daroczi, Borbala [Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania (United States); Xu Xia [Merck Research Laboratories, North Wales, Pennsylvania (United States); Dicker, Adam P. [Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania (United States)

    2012-01-01

    Purpose: The purpose of this study was to determine the ability of radiation therapy (RT) combined with the tyrosine kinase inhibitors (TKI) vandetanib (antiepidermal growth factor receptor [EGFR] plus antivascular endothelial growth factor receptor [anti-VEGFR]) and cediranib (anti-VEGFR) to inhibit glioblastoma multiforme (GBM) growth. A secondary aim was to investigate how this regimen is modulated by tumor EGFR expression. Methods and Materials: Radiosensitivity was assessed by clonogenic cell survival assay. VEGF secretion was quantified by enzyme-linked immunosorbent assay. GBM (U87MG wild-type EGFR [wtEGFR] and U87MG EGFR-null) xenografts were treated with vandetanib, cediranib, and RT, alone or in combinations. Excised tumor sections were stained for proliferative and survival biomarkers. Results: In vitro, U87MG wtEGFR and U87 EGFR-null cells had similar growth kinetics. Neither TKI affected clonogenic cell survival following RT. However, in vivo, exogenous overexpression of wtEGFR decreased tumor doubling time (T2x) in U87MG xenografts (2.70 vs. 4.41 days for U87MG wtEGFR vs. U87MG vector, respectively). In U87MG EGFR-null cells, TKI combined with radiation was no better than radiation therapy alone. In U87MG wtEGFR, RT in combination with vandetanib (but not with cediranib) significantly increased tumor T2x compared with RT alone (T2x, 10.4 days vs. 4.8 days; p < 0.001). In vivo, growth delay correlated with suppression of pAkt, survivin, and Ki67 expression in tumor samples. The presence of EGFR augmented RT-stimulated VEGF release; this effect was inhibited by vandetanib. Conclusions: EGFR expression promoted tumor growth in vivo but not in vitro, suggesting a microenvironmental effect. GBM xenografts expressing EGFR exhibited greater sensitivity to both cediranib and vandetanib than EGFR-null tumors. Hence EGFR status plays a major role in determining a tumor's in vivo response to radiation combined with TKI, supporting a &apos

  1. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

    OpenAIRE

    Gronwald, R G; Grant, F J; Haldeman, B A; Hart, C E; O'Hara, P J; Hagen, F S; Ross, R.; Bowen-Pope, D F; Murray, M. J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately ...

  2. Ceramic materials and growth factors

    Energy Technology Data Exchange (ETDEWEB)

    Ohgushi, H.; Yoshikawa, T.; Okumura, M.; Nakajima, H.; Takakura, Y. [Nara Medical Univ. (Japan). Dept. of Orhtopaedic Surgery; Dohi, Y. [Nara Medical Univ. (Japan). Dept. of Public Health; Noshi, T.; Ikeuchi, M. [Nara Medical Univ. (Japan). Dept. of Oral and Maxillofacial Surgery

    2001-07-01

    Recently, many types of growth factors have been purified and used for promoting cell differentiation cascade. The activity of growth factors can be detected in vitro such as culture condition. However, the activity is difficult to detect when these factors are locally administered in vivo, because these dissipate soon after the administration. In order to retain growth factors in local milieu, these can be incorporated with biocompatible porous ceramic materials. Such ceramic/factors composites when implanted in vivo, can trigger certain types of cell differentiation cascade resulted in new tissue formation and tissue regeneration. The paper describes the ceramic / growth factors composites especially hydroxyapatite ceramic (HA) / bone morphogenetic protein (BMP) composite to induce osteoblastic differentiation of mesenchymal stem cells. The HA/BMP composite supported the osteoblastic differentiation on the HA surface and finally resulted in bone bonding to the HA. When the marrow mesenchymal stem cells (MSCs) were impregnated in pore areas of HA ceramics, the composites showed more and rapid bone formation than the HA/BMP and HA/MSCs composite, indicating the synergistic effect of BMP and MSCs. These findings indicate the importance of ceramic surface to evoke osteoblastic differentiation as well as to capture the molecules of growth factors for the cell differentiation. (orig.)

  3. Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients.

    Science.gov (United States)

    Chomiski, Verônica; Gragnani, Alfredo; Bonucci, Jéssica; Correa, Silvana Aparecida Alves; Noronha, Samuel Marcos Ribeiro de; Ferreira, Lydia Masako

    2016-08-01

    To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

  4. Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor beta-induced protein (TGFBIp).

    Science.gov (United States)

    Runager, Kasper; García-Castellanos, Raquel; Valnickova, Zuzana; Kristensen, Torsten; Nielsen, Niels Chr; Klintworth, Gordon K; Gomis-Rüth, F Xavier; Enghild, Jan J

    2009-03-01

    Transforming growth factor beta-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported.

  5. Nerve growth factor promotes human sperm motility in vitro by increasing the movement distance and the number of A grade spermatozoa.