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Sample records for permeabilized fibroblasts effects

  1. Membrane Permeabilization Induced by Sphingosine: Effect of Negatively Charged Lipids

    PubMed Central

    Jiménez-Rojo, Noemi; Sot, Jesús; Viguera, Ana R.; Collado, M. Isabel; Torrecillas, Alejandro; Gómez-Fernández, J.C.; Goñi, Félix M.; Alonso, Alicia

    2014-01-01

    Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease. PMID:24940775

  2. Effects of inositol trisphosphate on calcium mobilization in high-voltage and saponin-permeabilized platelets

    SciTech Connect

    Gear, A.R.L.; Hallam, T.J.

    1986-03-01

    Interest in phosphatidylinositol metabolism has been greatly stimulated by the findings that diglyceride and inositol phosphates may serve as second messengers in modulating cellular function. Formation of 1,4,5-inositol trisphosphate (IP/sub 3/), in particular, has been linked to mobilization of intracellular calcium in a number of cell types. The authors have examined the ability of IP/sub 3/ to mobilize calcium in human platelets permeabilized by either saponin or high-voltage discharge. Saponin at 15 ..mu..g/ml effectively permeabilized platelets to exogenous inositol 1,4,5-trisphosphate which released bound (/sup 45/Ca) within 1 min and with a Ka of 7.4 +/- 4.1 ..mu..M. A small (25%) azide-sensitive pool was also responsive to inositol trisphosphate. The calcium pools were completely discharged by A-23187 and the ATP-dependent uptake was prevented by dinitrophenol. In contrast to the result with saponin, platelets accessed by high-voltage discharge were insensitive to challenge by inositol 1,4,5-trisphosphate. The data suggest that while inositol 1,4,5-trisphosphate can rapidly mobilize platelet calcium, the ability to demonstrate this depends on the method of permeabilization.

  3. Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons

    PubMed Central

    YAN, MIN; ZHU, WENBO; ZHENG, XIAOKE; LI, YUAN; TANG, LIPENG; LU, BINGZHENG; CHEN, WENLI; QIU, PENGXIN; LENG, TIANDONG; LIN, SUIZHEN; YAN, GUANGMEI; YIN, WEI

    2016-01-01

    Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK-801 and the calcium chelator ethylene glycolbis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal-associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α-fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N-Acetyl-L-cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity. PMID:26821268

  4. Permeabilizing biofilms

    DOEpatents

    Soukos, Nikolaos S.; Lee, Shun; Doukas, Apostolos G.

    2008-02-19

    Methods for permeabilizing biofilms using stress waves are described. The methods involve applying one or more stress waves to a biofilm, e.g., on a surface of a device or food item, or on a tissue surface in a patient, and then inducing stress waves to create transient increases in the permeability of the biofilm. The increased permeability facilitates delivery of compounds, such as antimicrobial or therapeutic agents into and through the biofilm.

  5. Effect of hydrogen peroxide and hypochlorite on mitochondrial membrane potential in permeabilized rat heart cells

    SciTech Connect

    Konno, N.; Kako, K.J. )

    1991-03-15

    The chemiosmotic theory states that the proton electrochemical potential gradient across the membrane drives mitochondrial energy transduction. Mitochondria can take up Ca accumulated in the cytosol. Therefore, oxidant-induced ATP depletion and Ca overload in the cell may be the result of mitochondrial dysfunction. Consequently, the authors measured membrane potential of mitochondria in situ in isolated rat heart myocytes with {sup 3}H-triphenylmethylphosphonium. This was followed by permeabilization using digitonin and rapid centrifugation using density gradient of bromododecane. They found that the membrane potentials, 118 mV with isolated and 161 mV with in situ mitochondria, were relatively well maintained under oxidant stress. High concentrations of oxidants reduced also the cellular ATP level, whereas the matrix volume was not significantly changed. The H{sub 2}O{sub 2} effect on the mitochondrial membrane potential was more pronounced when the extra-mitochondrial free Ca concentration was increased in permeabilized myocytes. These results support the view that heart mitochondria are equipped with well developed defense mechanisms against oxidants and thus the electrochemical gradient of inner membrane is affected only by a relatively large concentration of H{sub 2}O{sub 2} and HOCl.

  6. Permeabilization of plant tissues by monopolar pulsed electric fields: effect of frequency.

    PubMed

    Asavasanti, Suvaluk; Ristenpart, William; Stroeve, Pieter; Barrett, Diane M

    2011-01-01

    Pulsed electric fields (PEF) nonthermally induce cell membrane permeabilization and thereby improve dehydration and extraction efficiencies in food plant materials. Effects of electrical field strength and number of pulses on plant tissue integrity have been studied extensively. Two previous studies on the effect of pulse frequency, however, did not provide a clear view: one study suggested no effect of frequency, while the other found a greater impact on tissue integrity at lower frequency. This study establishes the effect of pulse frequency on integrity of onion tissues. Changes in electrical characteristics, ion leakage, texture parameters, and percent weight loss were quantified for a wide range of pulse frequencies under conditions of fixed field strength and pulse number. Optical microscopy and viable-cell staining provided direct visualization of effects on individual cells. The key finding is that lower frequencies (f < 1 Hz) cause more damage to tissue integrity than higher frequencies (f = 1 to 5000 Hz). Intriguingly, the optical microscopy observations demonstrate that the speed of intracellular convective motion (that is, cytoplasmic streaming) following PEF application is strongly correlated with PEF frequency. We provide the first in situ visualization of the intracellular consequence of PEF at different frequencies in a plant tissue. We hypothesize that cytoplasmic streaming plays a significant role in moving conductive ionic species from permeabilized cells to the intercellular space between plant cells, making subsequent pulses more efficacious at sufficiently low frequencies. The results suggest that decreasing the pulse frequency in PEF may minimize the number of pulses needed to achieve a desired amount of permeabilization, thus lowering the total energy consumption. Practical Application: PEF cause pores to be formed in plant cell membranes, thereby improve moisture removal and potential extraction of desirable components. This study used in

  7. Permeabilized myocardial fibers as model to detect mitochondrial dysfunction during sepsis and melatonin effects without disruption of mitochondrial network.

    PubMed

    Doerrier, Carolina; García, José A; Volt, Huayqui; Díaz-Casado, María E; Luna-Sánchez, Marta; Fernández-Gil, Beatriz; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-03-01

    Analysis of mitochondrial function is crucial to understand their involvement in a given disease. High-resolution respirometry of permeabilized myocardial fibers in septic mice allows the evaluation of the bioenergetic system, maintaining mitochondrial ultrastructure and intracellular interactions, which are critical for an adequate functionality. OXPHOS and electron transport system (ETS) capacities were assessed using different substrate combinations. Our findings show a severe septic-dependent impairment in OXPHOS and ETS capacities with mitochondrial uncoupling at early and late phases of sepsis. Moreover, sepsis triggers complex III (CIII)-linked alterations in supercomplexes structure, and loss of mitochondrial density. In these conditions, melatonin administration to septic mice prevented sepsis-dependent mitochondrial injury in mitochondrial respiration. Likewise, melatonin improved cytochrome b content and ameliorated the assembly of CIII in supercomplexes. These results support the use of permeabilized fibers to identify properly the respiratory deficits and specific melatonin effects in sepsis. PMID:26748191

  8. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5

    PubMed Central

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  9. EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

    PubMed

    Zhang, Qi; Wang, Decheng; Jiang, Guozhong; Liu, Wei; Deng, Qing; Li, Xiujun; Qian, Wei; Ouellet, Hugues; Sun, Jianjun

    2016-01-01

    EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation. PMID:27600772

  10. The effect of Tween 80 on eggshell permeabilization in Galleria mellonella (L.) (Lepidoptera, Pyralidae).

    PubMed

    Cosi, E; Abidalla, M T; Roversi, P F

    2010-01-01

    The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane. PMID:20818457

  11. Effect of electrode geometry on field strength in plastic microfluidic devices and application to cell membrane permeabilization

    NASA Astrophysics Data System (ADS)

    Chooljian, Marc; Paredes, Jacobo; Liepmann, Dorian

    2014-11-01

    We have developed a method that allows embedding of electrodes in up to 3 walls of a plastic microfluidic channel. Electric field strength and homogeneity of various electrode geometries is analyzed theoretically and experimentally by evaluating the efficiency of on-chip lysis of cells. Electric field-mediated disruption of membranes is an important tool in diagnostics, basic biology, and synthetic biology due to the ability to permeabilize the cell membrane without changing the chemical composition of the buffer. Typically, fields of the required magnitude are applied to the cell by discharging a capacitor through a mixture of cells in a cuvette, resulting in a transient high-voltage pulse. We demonstrate that is possible to substitute a spatially varied DC electric field along a microchannel and to control the timing of the pulses by changing the electrode spacing and the flow rate. Homogeneity of the field with respect to the cross section of the channel is key to achieving critical field strength regardless of the cell's lateral position in the channel. A comparison of 2D versus 3D electrode geometries on the efficiency of electroporation and on side-effects arising due to the electric field (recirculating flows and hydrolysis) is presented.

  12. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles.

    PubMed Central

    Rinaldi, Andrea C; Mangoni, Maria Luisa; Rufo, Anna; Luzi, Carla; Barra, Donatella; Zhao, Hongxia; Kinnunen, Paavo K J; Bozzi, Argante; Di Giulio, Antonio; Simmaco, Maurizio

    2002-01-01

    The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings. PMID:12133008

  13. Effect of pulsed electric field treatments on permeabilization and extraction of pigments from Chlorella vulgaris.

    PubMed

    Luengo, Elisa; Condón-Abanto, Santiago; Álvarez, Ignacio; Raso, Javier

    2014-12-01

    The effect of pulsed electric field (PEF) treatments of different intensities on the electroporation of the cytoplasmatic membrane of Chlorella vulgaris, and on the extraction of carotenoids and chlorophylls were investigated. Staining the cells with propidium iodide before and after the PEF treatment revealed the existence of reversible and irreversible electroporation. Application of PEF treatments in the range of 20-25 kV cm(-1) caused most of the population of C. vulgaris to be irreversibly electroporated even at short treatment times (5 pulses of 3 µs). However, at lower electric field strengths (10 kV cm(-1)), cells that were reversibly electroporated were observed even after 50 pulses of 3 µs. The electroporation of C. vulgaris cells by PEF higher than 15 kV cm(-1) and duration is higher than 15 µs increased significantly the extraction yield of intracellular components of C. vulgaris. The application of a 20 kV cm(-1) for 75 μs increased the extraction yield just after the PEF treatment of the carotenoids, and chlorophylls a and b 0.5, 0.7, and 0.8 times, respectively. However, further increments in electric field strength and treatment time did not cause significant increments in the extraction yield. The extraction of carotenoids from PEF-treated C. vulgaris cells after 1 h of the application of the treatment significantly increased the extraction yield in comparison to the yield obtained from the cells extracted just after the PEF treatment. After PEF treatment at 20 kV cm(-1) for 75 µs, extraction yield for carotenoids, and chlorophylls a and b increased 1.2, 1.6, and 2.1 times, respectively. A high correlation was observed between irreversible electroporation and percentage of yield increase when the extraction was conducted after 1 h of the application of PEF treatment (R: 0.93), but not when the extraction was conducted just after PEF treatment (R: 0.67). PMID:24880235

  14. Excision of ultraviolet damage and the effect of irradiation on DNA synthesis in a strain of Bloom's syndrome fibroblasts

    SciTech Connect

    Henson, P.; Selsky, C.A.; Little, J.B.

    1981-03-01

    Researchers have studied repair of ultraviolet light-induced damage in a strain of Bloom's syndrome cells which we have shown to be defective in host cell reactivation of uv-irradiated herpes simplex virus. Excision repair was monitored by following loss of sensitivity of DNA in permeabilized cells to digestion by the Micrococcus luteus uv endonuclease preparation. The Bloom's syndrome fibroblasts apparently removed endonuclease-sensitive sites from the DNA slightly less efficiently than did normal strains. After 24 h, 38% of the sites remained in the Bloom's syndrome cells in comparison with 16% in normal fibroblasts. DNA newly synthesized in uv-irradiated Bloom's syndrome cells sedimented less far into alkaline sucrose gradients than did DNA from similarly treated normal cells. In other respects, including the effect of caffeine exposure, DNA synthesis in Bloom's syndrome cells was indistinguishable from that in normal cells. We were therefore able to detect only minor defects in the repair of uv-induced damage in Bloom's syndrome fibroblasts. This is consistent with the normal survival exhibited by these cells. The defect in excision repair may, however, be sufficient to allow the cellular repair capacity to become saturated at high infecting multiplicities of uv-irradiated herpes simplex virus.

  15. The antifibrosis effect of adrenomedullin in human lung fibroblasts.

    PubMed

    Hao, Shu-Ling; Yu, Zhong-He; Qi, Bao-Shen; Luo, Ji-Zheng; Wang, Wei-Ping

    2011-12-01

    Adrenomedullin (AM) is a regulatory peptide involved in cellular proliferation and protein synthesis. The authors investigated AM and the AM receptor system in the human fetal lung fibroblasts (HFLFs), and assessed whether AM can inhibit proliferation and collagen synthesis in HFLFs under hypoxia. Fibroblasts were exposed to hypoxia (2% O(2)) after the addition of AM. The effects of AM and transforming growth factor β1 (TGF-β1) on the proliferation of fibroblasts were determined by the methanethiosulfonate (MTS) assay. Total collagen synthesis was determined by [(3)H]proline incorporation. TGF-β1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of intracellular calciumion ([Ca(2+)](i)) in fibroblasts was detected with a laser scanning confocal microscope. AM, adrenomedullin receptor (ADMR), calcitonin receptor-like receptor (CRLR), AM receptor chaperone receptor activity-modifying protein-1 (RAMP1),RAMP2, and RAMP3 were detected in the HFLFs. The hypoxia-induced increases in cell proliferation, collagen synthesis, and TGF-β1 production were inhibited by AM. AM also inhibited proliferation and collagen synthesis in fibroblasts induced by TGF-β1. AM caused a decrease of the hypoxia-induced [Ca(2+)](i) in fibroblasts. This study suggests that AM is produced by HFLFs and AM may function as an antifibrosis factor that protects cells from hypoxic pulmonary damage through its receptors. PMID:22087514

  16. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    PubMed Central

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD. PMID:27057276

  17. Anti-fibrotic effects of theophylline on lung fibroblasts

    SciTech Connect

    Yano, Yukihiro; Yoshida, Mitsuhiro . E-mail: hiroinosaka@hotmail.com; Hoshino, Shigenori; Inoue, Koji; Kida, Hiroshi; Yanagita, Masahiko; Takimoto, Takayuki; Hirata, Haruhiko; Kijima, Takashi; Kumagai, Toru; Osaki, Tadashi; Tachibana, Isao; Kawase, Ichiro

    2006-03-17

    Theophylline has been used in the management of bronchial asthma and chronic obstructive pulmonary disease for over 50 years. It has not only a bronchodilating effect, but also an anti-inflammatory one conducive to the inhibition of airway remodeling, including subepithelial fibrosis. To date however, whether theophylline has a direct inhibitory effect on airway fibrosis has not been established. To clarify this question, we examined whether theophylline affected the function of lung fibroblasts. Theophylline suppressed TGF-{beta}-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation stimulated by FBS and TGF-{beta}-induced {alpha}-SMA protein. A cAMP analog also inhibited TGF-{beta}-induced COL1 mRNA expression in lung fibroblasts. A PKA inhibitor reduced the inhibitory effect of theophylline on TGF-{beta}-induced COL1 mRNA expression. These results indicate that theophylline exerts anti-fibrotic effects, at least partly, through the cAMP-PKA pathway.

  18. Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

    PubMed Central

    Wang, Xuan; Fan, Ya-Zhi; Yao, Liang; Wang, Jian-Ming

    2016-01-01

    AIM To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon's capsule, both in vitro and in vivo. METHODS Human primary Tenon's capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT) method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1,2) and proliferating cell nuclear antigen (PCNA). Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d). A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson's trichrome to compare the neovascularization in the subconjunctiva area. RESULTS In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson's trichrome observation. CONCLUSION By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery. PMID:27275419

  19. The effects of acoustic vibration on fibroblast cell migration.

    PubMed

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. PMID:27612824

  20. Effect of Phenytoin and Age on Gingival Fibroblast Enzymes

    PubMed Central

    Vahabi, Surena; Nazemisalman, Bahareh; Vahid Golpaigani, Mojtaba; Ahmadi, Anahid

    2014-01-01

    Objective: The alteration of cytokine balance is stated to exert greater influence on gingival overgrowth compared to the direct effect of the drug on the regulation of extracellular matrix metabolism. The current study evaluated the effect of phenytoin on the regulation of collagen, lysyl oxidase and elastin in gingival fibroblasts. Materials and Methods: Normal human gingival fibroblasts (HGFs) were obtained from 4 healthy children and 4 adults. Samples were cultured with phenytoin. MTT test was used to evaluate the proliferation and ELISA was performed to determine the level of IL1β and PGE2 production by HGFs. Total RNA of gingival fibroblasts was extracted and RT-PCR was performed on samples. Mann-Whitney U test was used to analyze the data with an alpha error level less than 0.05. Results: There was a significant difference in the expression of elastin between the controls and treated samples in both adult and pediatric groups and also in the lysyl oxidase expression of adult controls and treated adults. No significant difference was found between collagen expression in adults. Conclusion: The significant difference in elastin and lysyl oxidase expression between adult and pediatric samples indicates the significant effect of age on their production. PMID:25628662

  1. Effects of metal ions on fibroblasts and spiral ganglion cells.

    PubMed

    Paasche, G; Ceschi, P; Löbler, M; Rösl, C; Gomes, P; Hahn, A; Rohm, H W; Sternberg, K; Lenarz, T; Schmitz, K-P; Barcikowski, S; Stöver, T

    2011-04-01

    Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 μmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated. PMID:21312225

  2. Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins.

    PubMed Central

    Sloan, D C; Haslam, R J

    1997-01-01

    Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane

  3. The Effect of Gynostemma pentaphyllum Extract on Mouse Dermal Fibroblasts

    PubMed Central

    Lobo, Sara Nadia; Qi, Yu Qing; Liu, Quan Zhong

    2014-01-01

    Background. The objective of this paper is to demonstrate the effect of Gynostemma pentaphyllum extract on mouse dermal fibroblasts. Recent studies have shown that this plant may possess great antioxidant properties, which can be very beneficial in combating oxidative stress. Methods. Gynostemma pentaphyllum extract was prepared and mouse dermal fibroblasts were obtained and cultured as per our laboratory protocols. Twelve samples of cells were cultured under the same conditions and both negative and positive controls were established. Induction of oxidative stress was carried out using ultraviolet C (UVC) light. Viable cell count was carried out, using microscopy. The analysis of the overall results was processed using SPSS version 16.0. Results. Statistical analysis showed strong positive correlation between the concentration of Gynostemma pentaphyllum and the mean duration of cell viability (rs = 1), with a high level of statistical significance (P < 0.01). Likewise, strong positive correlation existed between trials of cell viability (rs = 0.988–1), with statistical significance (P < 0.01). Conclusion. Gynostemma pentaphyllum extract prolongs viability of mouse dermal fibroblasts damaged by UVC light-induced oxidative stress. The results show the potential benefits of this extract on dermal cell aging. PMID:24729883

  4. The Effect of Gynostemma pentaphyllum Extract on Mouse Dermal Fibroblasts.

    PubMed

    Lobo, Sara Nadia; Qi, Yu Qing; Liu, Quan Zhong

    2014-01-01

    Background. The objective of this paper is to demonstrate the effect of Gynostemma pentaphyllum extract on mouse dermal fibroblasts. Recent studies have shown that this plant may possess great antioxidant properties, which can be very beneficial in combating oxidative stress. Methods. Gynostemma pentaphyllum extract was prepared and mouse dermal fibroblasts were obtained and cultured as per our laboratory protocols. Twelve samples of cells were cultured under the same conditions and both negative and positive controls were established. Induction of oxidative stress was carried out using ultraviolet C (UVC) light. Viable cell count was carried out, using microscopy. The analysis of the overall results was processed using SPSS version 16.0. Results. Statistical analysis showed strong positive correlation between the concentration of Gynostemma pentaphyllum and the mean duration of cell viability (rs = 1), with a high level of statistical significance (P < 0.01). Likewise, strong positive correlation existed between trials of cell viability (rs = 0.988-1), with statistical significance (P < 0.01). Conclusion. Gynostemma pentaphyllum extract prolongs viability of mouse dermal fibroblasts damaged by UVC light-induced oxidative stress. The results show the potential benefits of this extract on dermal cell aging. PMID:24729883

  5. Effect of microemulsions on cell viability of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  6. Effect of Arctium lappa (burdock) extract on canine dermal fibroblasts.

    PubMed

    Pomari, Elena; Stefanon, Bruno; Colitti, Monica

    2013-12-15

    Although the biological activities of Arctium lappa (burdock) have been already investigated in human and other species, data evaluating the molecular mechanisms have not been reported in the dog. In this study we analyzed for the first time the effect of a root extract of burdock on molecular responses in canine dermal fibroblasts with H2O2 stimulation (H group), with burdock treatment (B group) and with H2O2 stimulation and burdock treatment (BH group), using RNAseq technology. Differentially expressed genes (P<0.05) of H, B and BH groups in comparison to the untreated sample (negative control, C group) were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). The expression profile of canine dermal fibroblasts treated with burdock extract with or without H2O2 stimulation, showed an up-regulation of mitochondrial superoxide dismutase (SOD2), disheveled 3 (DVL3) and chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGALNACT2). The data suggested that burdock has implications in cell adhesion and gene expression with the modulation of Wnt/β catenin signaling and Chondroitin Sulphate Biosynthesis that are particularly important for the wound healing process. PMID:24192279

  7. Protective effect of pantothenic acid and related compounds against permeabilization of Ehrlich ascites tumour cells by digitonin.

    PubMed

    Slyshenkov, V S; Rakowska, M; Wojtczak, L

    1996-01-01

    Preincubation of Ehrlich ascites tumour cells with millimolar concentrations of pantothenic acid, pantothenol or pantethine, but not with homopantothenic acid, at 22 degrees C or 32 degrees C, but not at 0 degrees C, makes the plasma membrane more resistant to the damaging effect of submillimolar concentrations of digitonin. It is proposed that this increased resistance is due to the increased rate of cholesterol biosynthesis. In fact, incorporation of [14C]acetate into cholesterol is by 45% increased in the cells preincubated with pantothenic acid; this probably reflects elevation of the content of CoA in such cells [Slyshenkov, V.S., Rakowska, M., Moiseenok, A.G. & Wojtczak, L. (1995) Free Radical Biol. Med. 19, 767-772]. PMID:8862188

  8. Effect of storage media on the proliferation of periodontal ligament fibroblasts

    SciTech Connect

    Lauer, H.C.; Mueller, J.G.; Gross, J.; Horster, M.F.

    1987-07-01

    The effect of storage media, which are routinely used in replantation, upon the proliferative capacity of periodontal ligament fibroblasts, was compared with the effect of a tissue culture medium. The periodontal tissue was obtained from mandibular central incisors of White New Zealand rabbits. The experiments were performed in fibroblasts derived during second subculture. The storage media were physiologic salt solution, Ringer's solution and Rivanol; the tissue culture medium was alpha-minimum essential medium without nucleosides. The incubation period was 1 hour. (/sup 3/H)-thymidine incorporation and cell counts were taken to indicate changes in the proliferative capacity of the fibroblasts. The tissue culture experiments showed that the proliferative ability of the periodontal ligament fibroblasts was dependent upon the composition of the storage medium. Physiologic salt solution, Ringer's solution and Rivanol were unable to maintain the metabolism of the fibroblasts. alpha-MEM medium, however, was capable of stimulating proliferation of the periodontal ligament fibroblasts.

  9. The effect of ciprofloxacin on tendon, paratenon, and capsular fibroblast metabolism.

    PubMed

    Williams, R J; Attia, E; Wickiewicz, T L; Hannafin, J A

    2000-01-01

    The pathologic mechanisms underlying fluoroquinolone-induced tendinopathy are poorly understood. The observed incidence of tendinitis and tendon rupture in patients treated with ciprofloxacin hydrochloride suggests that the fluoroquinolone antibiotics alter tendon fibroblast metabolism. The purpose of this study was to examine the effect of ciprofloxacin on fibroblast metabolism in vitro. Canine Achilles tendon, paratenon, and shoulder capsule specimens were maintained in culture with ciprofloxacin (5, 10, or 50 microg/ml). Fibroblast proliferation, collagen synthesis, proteoglycan synthesis, and matrix-degrading activity were analyzed. Incubation of Achilles tendon, Achilles paratenon, and shoulder capsule fibroblasts with ciprofloxacin resulted in a statistically significant 66% to 68% decrease in cell proliferation compared with control cells at day 3 in culture. Ciprofloxacin caused a statistically significant 36% to 48% decrease in collagen synthesis compared with controls in all fibroblast cultures. Ciprofloxacin caused a statistically significant 14% to 60% decrease in proteoglycan synthesis in all fibroblast cell lines. Compared with unstimulated control fibroblasts, culture media from Achilles tendon, paratenon, and shoulder capsule cells that were exposed to ciprofloxacin demonstrated statistically significant increases in matrix-degrading proteolytic activity after 72 hours in culture. This study demonstrates that ciprofloxacin stimulates matrix-degrading protease activity from fibroblasts and that it exerts an inhibitory effect on fibroblast metabolism. The increase in protease activity and the inhibition of both cell proliferation and the synthesis of matrix ground substance may contribute to the clinically described tendinopathies associated with ciprofloxacin therapy. PMID:10843129

  10. Effect of lipopolysaccharide on the biological characteristics of human skin fibroblasts and hypertrophic scar tissue formation.

    PubMed

    Yang, Hongming; Hu, Chao; Li, Fengyu; Liang, Liming; Liu, Lingying

    2013-06-01

    Burn injury-mediated destruction of the skin barrier normally induces microbial invasion, in turn leading to the development of systemic infection and occasional septic shock by the release of endotoxins. The objective of this work was to study the influence of lipopolysaccharide (LPS) on the biological characteristics of normal skin fibroblasts and to elucidate the influence of LPS in the initial stage of skin wound healing. Twenty patients with hypertrophic scar in proliferative stage were selected randomly and primary cultures were established from fibroblasts derived from their hypertrophic scar tissue and normal skin. Normal skin fibroblasts of passage 3 were stimulated with different concentrations of LPS. LPS stimulated the proliferation and collagen synthesis of fibroblasts within a certain extent of concentrations (0.005-0.5 μg/mL) (P < 0.05), whereas at a concentration of 1 μg/mL inhibited the proliferation and collagen synthesis of fibroblasts (P < 0.05). Collagen synthesis by normal skin fibroblasts after LPS stimulation mimicked those derived from hypertrophic scar tissue. LPS of 0.1 μg/mL had significant effect on normal skin fibroblasts-continuous passage of these fibroblasts resulted in ultrastructural pattern similar to fibroblasts derived from hypertrophic scar tissue, and the findings was substantiated by hematoxylin and eosin staining and immunohistochemistry detection of proliferation cell nuclear antigen, type I procollagen and α-smooth muscle actin. Our results suggest that LPS might convert normal skin fibroblasts to hypertrophic scar tissue fibroblasts and participate in the formation of hypertrophic scar; hence, appropriate concentration of LPS may have no effect or be beneficial to skin wound healing, whereas excessive concentration of LPS may delay the time of wound healing. PMID:23653386

  11. In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes[S

    PubMed Central

    Ensenauer, Regina; Fingerhut, Ralph; Schriever, Sonja C.; Fink, Barbara; Becker, Marc; Sellerer, Nina C.; Pagel, Philipp; Kirschner, Andreas; Dame, Torsten; Olgemöller, Bernhard; Röschinger, Wulf; Roscher, Adelbert A.

    2012-01-01

    Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO. PMID:22345709

  12. The effect of phorbols on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of (/sup 3/H)hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total (/sup 3/H)hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of (/sup 3/H)nucleotides and is an indication of an interference with intercellular communication.

  13. Modulation of the effects of alveolar macrophages on lung fibroblast collagen production rate

    SciTech Connect

    Clark, J.G.; Greenberg, J.

    1987-01-01

    Alveolar macrophages (AM) may function as effector cells that can either stimulate or inhibit lung fibroblast collagen production. However, conditions that determine the predominant effect of AM on fibroblasts are not well understood. To delineate factors that modulate the effects of AM on lung fibroblasts, we studied the interaction of AM products and fibroblasts in vitro. The AM were obtained by bronchoalveolar lavage of hamsters with bleomycin-induced pulmonary fibrosis. Conditioned medium (CM) from the AM cultures was incubated in varying amounts with lung fibroblast (IMR-90) cultures. After metabolic labeling with (/sup 3/H)proline, fibroblast collagen production based on procollagen-specific radioactivity was determined. Macrophage CM in concentrations greater than 5% suppressed collagen production, an event attributed to the macrophage-derived suppressive factor that we have previously characterized. Macrophages were also determined to produce PGE2 in culture. Authentic PGE2 at concentrations found in CM was found to suppress fibroblast collagen production, indicating that AM-derived PGE2 contributes to the suppressive activity in CM. To examine possible stimulatory factors in CM, the fibroblasts were preincubated with indomethacin. This approach was based on our previous observation that AM-derived suppressive factor increases endogenous fibroblast PGE2 and that its activity can be blocked by indomethacin. Macrophage CM in a concentration of 20% did not suppress the collagen production of indomethacin-treated fibroblasts. However, CM concentrations of 5 and 10% increased collagen production (173 and 143% of control values, respectively), indicating the presence of stimulatory factor(s) in macrophage-conditioned medium.

  14. Paracrine Effects of Adipose-Derived Stem Cells on Keratinocytes and Dermal Fibroblasts

    PubMed Central

    Lee, Seung Ho; Jin, Sang Yun; Song, Jin Seok; Seo, Kyle K.

    2012-01-01

    Background Adipose-derived stem cells (ASCs) are mesenchymal stem cells that have recently been applied to tissue repair and regeneration. Keratinocytes and dermal fibroblasts play key roles in cutaneous wound healing. Objective We investigated the paracrine effects of ASCs on HaCaT cells (i.e., immortalized human keratinocytes) and human dermal fibroblasts to explore the mechanism of the effects of ASCs on cutaneous wound healing. Methods HaCaT cells and primary cultured human dermal fibroblasts were treated with 50% conditioned medium of ASCs (ASC-CM). Viability, in vitro wound healing, and fibroblast-populated collagen lattice contraction assays were conducted, and reverse transcription-polymerase chain reaction (RT-PCR) for the type I procollagen α1 chain gene was performed. Results The proliferation of HaCaT cells and fibroblasts was increased by ASC-CM in the viability assay. ASC-CM promoted in vitro wound healing of HaCaT cells and increased the contraction of the fibroblast-populated collagen lattice. RT-PCR showed that the transcription of the type I procollagen α1 chain gene in fibroblasts was upregulated by ASC-CM. Conclusion The stimulatory effect of ASC on cutaneous wound healing may be partially mediated by paracrine effects of ASCs on other skin cells. Application of ASCs or ASC-derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions. PMID:22577262

  15. Osteostimulatory effect of bone grafts on fibroblast cultures

    PubMed Central

    Fathima, Hameed; Harish

    2015-01-01

    Objective: We analyzed the morphological changes and alkaline phosphatase (ALP) level in fibroblast, which is indicative of their functional ability when cultured in three different commercially available graft materials with osseoconductive property. Materials and Methods: Fibroblasts obtained from fifth passage were seeded within three different bone substitutes (bovine hydroxyapatite [HA] [Osseo-graft®], β-tricalciumphosphate [RTR®], bovine HA [Bio-oss®]) and incubated under standard cell culture conditions. 10 samples in each group were evaluated for cell morphology and alkaline phosphates activity using scanning electron microscopy and spectrophotometric analysis on the 7th day of culture. Results: Fibroblast cultured with RTR® showed changes in morphology and increase in ALP activity when compared to fibroblast cultured with Osseo-graft® and Bio-oss®. Conclusion: Alkaline phosphatase activity was observed in fibroblasts when cultured with three types of commercially available bone grafts. ALP activity was highest when cultured with β-tricalcium phosphate graft material indicating its better bone regenerating capacity of this graft material. PMID:26283815

  16. Mutagenic effects of alpha particles in normal human skin fibroblasts

    SciTech Connect

    Chen, D.J.; Carpenter, S.; Hanks, T.

    1992-12-31

    Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV {mu}m{sup {minus}1}) emitted from the decay of {sup 238}Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

  17. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    PubMed

    Parker, William H; Qu, Zhi-chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  18. Irradiated fibroblast-induced bystander effects on invasive growth of squamous cell carcinoma under cancer-stromal cell interaction.

    PubMed

    Kamochi, Noriyuki; Nakashima, Masahiro; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Sugihara, Hajime; Toda, Shuji; Kudo, Sho

    2008-12-01

    The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved. PMID:19018771

  19. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  20. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

    PubMed

    Varma, Sandeep R; Sivaprakasam, Thiyagarajan O; Mishra, Abheepsa; Kumar, L M Sharath; Prakash, N S; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  1. Biological effects of near-infrared lasers on fibroblast cellular differentiation, proliferation and contraction

    NASA Astrophysics Data System (ADS)

    Acquaviva, Joseph T.; Chen, Wei R.; Vaughan, Melville B.

    2013-02-01

    Combining near infrared (NIR) laser irradiation into a tumor treatment therapy has shown promising results. For a comprehensive tumor therapy, it is important to understand the effects of NIR irradiation not only on the tumor, but on the tumor stroma as well. The composition of the microenvironment present near the tumor cells is critical to the phenotype of the tumor. Fibroblasts affect tissue homeostasis and change the microenvironment surrounding the tumor. Myofibroblast are derived from fibroblast cells, and in some cases indicate the transformation of healthy tissue into malignant tissue. Wound healing environments are rich in fibroblast cells and are similar to tumor stromas. To simulate a tumor stroma a wound healing environment was constructed. Two different human fibroblast cells were cultured in collagen lattices. Specifically, collagen lattices were created, with type 1 collagen, incubated for 5 days and irradiated with a 980nm laser on the 4th day. The subsequent collagen lattices were either released and measured, or fixed for immunostaining on the 5th day; the contraction rates also were analyzed. Furthermore, collagen lattices were stained to identify fibroblast proliferation and differentiation, into myofibroblasts. The results suggested NIR laser irradiation had some biological effects on the fibroblast cells, but the full extent of the effects is still unclear.

  2. Strategies for Assaying Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Hafner Česen, Maruša; Turk, Boris

    2016-01-01

    Late endosomal organelles have an acidic pH and contain hydrolytic enzymes to degrade cargo delivered either from the extracellular environment by endocytosis or from within the cell itself by autophagy. In the event of lysosomal membrane permeabilization (LMP), the contents of late endosomes and lysosomes can be released into the cytosol and then initiate apoptosis. Compounds that can trigger LMP are therefore candidates for the induction of apoptosis, in particular in anticancer therapy. Alternatively, drug-delivery systems, such as nanoparticles, can have side effects that can include LMP, which has toxic consequences for the cells. To determine when, to what extent, and with what consequences LMP occurs is therefore of paramount importance for the evaluation of new potentially LMP-inducing compounds. In this introduction, we provide an overview of some basic assays for assessing LMP, such as staining with lysosomotropic dyes and measurement of cysteine cathepsin activity, and discuss additional strategies for the detection of the release of endogenous lysosomal molecules or preloaded exogenous tracers into the cytosol. PMID:27250949

  3. Optically absorbing nanoparticle mediated cell membrane permeabilization.

    PubMed

    Bhattacharyya, Kiran; Mehta, Smit; Viator, John

    2012-11-01

    Membrane permeabilization is imperative for gene and drug delivery systems, along with other cell manipulation methods, since the average eukaryotic cell membrane is not permeable to polar and large nonpolar molecules. Antibody conjugated optically absorbing gold nanospheres are targeted to the cell membrane of T47D breast cancer cell line and irradiated with 5 ns pulse, 20 Hz, 532 nm light to increase membrane permeability. Up to 90% permeabilization with less than 6% death is reported at radiant exposures up to 10 times lower than those of other comparable studies. PMID:23114334

  4. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro

    PubMed Central

    Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology. PMID:27536196

  5. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology. PMID:27536196

  6. Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

    SciTech Connect

    Benamer, Najate; Bois, Patrick

    2011-04-29

    Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

  7. Effects of selenium on UVA-induced lipid peroxidation in cultured human skin fibroblasts.

    PubMed

    Moysan, A; Morlière, P; Marquis, I; Richard, A; Dubertret, L

    1995-01-01

    The effect of selenium on the lethal action of ultraviolet radiations and on the lipid peroxidation induced by exposures to ultraviolet A (320-400 nm; 360 kJ.m-2) and ultraviolet B (290-320 nm; 2 kJ.m-2) have been measured in cultured human skin fibroblasts. The experiments have been performed with either pure selenium or a spring water containing selenium and other trace elements (zinc and strontium). For cells cultured in a standard medium containing 10% fetal calf serum, no effect of selenium or spring water addition to the culture medium was observed on the lethality or on the peroxidative process induced by ultraviolet A and B radiations. Concurrently, there was no detectable increase of the seleno-dependent glutathione peroxidase activity. For cells previously depleted in selenium by a culture in a medium containing only 2% serum, a protective effect of selenium can be detected. Depending on the fibroblast donor, we observed (1) a protective effect on lethality of dividing fibroblasts induced by ultraviolet A radiations, (2) a protective effect on lipid peroxidation induced by ultraviolet A radiations on dividing or quiescent fibroblasts and (3) an increase in glutathione peroxidase activity in fibroblasts. PMID:7632435

  8. Biological Effects of Extracorporeal Shock Waves on Fibroblasts. A Review

    PubMed Central

    Frairia, Roberto; Berta, Laura

    2011-01-01

    Summary Tissue homeostasis is influenced by mechanical forces which regulate the normal function of connective tissues. Mechanotransduction, the process that transforms mechanical stimuli in chemical signals, involves mechanosensory units integrated in cell membrane. The mechanosensory units are able to activate gene expression for growth factors or cytochines as well as to induce a biological event which results in cell proliferation and/or differentiation. In connective tissue the fibroblasts are the cells more represented and are considered as a model of mechanosensitive cells. They are ubiquitous but specific for each type of tissue. Their heterogeneity consists in different morphological features and activity; the common function is the mechanosensitivity, the capacity to adhere to extracellular matrix (ECM) and to each other, the secretion of growth factors and ECM components. Extracorporeal shock waves (ESW) have been recently used to treat damaged osteotendineous tissues. Studies in vitro and in vivo confirmed that ESW treatment enhances fibroblast proliferation and differentiation by activation of gene expression for transforming growth factor β1 (TGF- β1) and Collagen Types I and III. In addition, an increase of nitric oxide (NO) release is even reported in early stage of the treatment and the subsequent activation of endothelial nitric oxide synthase (eNOS) and of vascular endothelial growth factor (VEGF) are related to TGF- β1 rise. The data have been related to the increase of angiogenesis observed in ESW treated tendons, an additional factor in accelerating the repairing process. A suitable treatment condition, characterized by a proper energy/shot number ratio, is the basis of treatment efficacy. Further ESWT applications are suggested in regenerative medicine, in all cases where fibroblast activity and the interaction with connective tissue can be positively influenced. PMID:23738262

  9. A Comparative Study on the Effects of Millisecond- and Microsecond-Pulsed Electric Field Treatments on the Permeabilization and Extraction of Pigments from Chlorella vulgaris.

    PubMed

    Luengo, Elisa; Martínez, Juan Manuel; Coustets, Mathilde; Álvarez, Ignacio; Teissié, Justin; Rols, Marie-Pierre; Raso, Javier

    2015-10-01

    The interdependencies of the two main processing parameters affecting "electroporation" (electric field strength and pulse duration) while using pulse duration in the range of milliseconds and microseconds on the permeabilization, inactivation, and extraction of pigments from Chlorella vulgaris was compared. While irreversible "electroporation" was observed above 4 kV/cm in the millisecond range, electric field strengths of ≥10 kV/cm were required in the microseconds range. However, to cause the electroporation of most of the 90 % of the population of C. vulgaris in the millisecond (5 kV/cm, 20 pulses) or microsecond (15 kV/cm, 25 pulses) range, the specific energy that was delivered was lower for microsecond treatments (16.87 kJ/L) than in millisecond treatments (150 kJ/L). In terms of the specific energy required to cause microalgae inactivation, treatments in the microsecond range also resulted in greater energy efficiency. The comparison of extraction yields in the range of milliseconds (5 kV, 20 ms) and microseconds (20, 25 pulses) under the conditions in which the maximum extraction was observed revealed that the improvement in the carotenoid extraction was similar and chlorophyll a and b extraction was slightly higher for treatments in the microsecond range. The specific energy that was required for the treatment in the millisecond range (150 kJ/L) was much higher than those required in the microsecond range (30 kJ/L). The comparison of the efficacy of both types of pulses on the extraction enhancement just after the treatment and after a post-pulse incubation period seemed to indicate that PEF in the millisecond range created irreversible alterations while, in the microsecond range, the defects were a dynamic structure along the post-pulse time that caused a subsequent increment in the extraction yield. PMID:25819916

  10. Effects of Current Provisional Restoration Materials on the Viability of Fibroblasts

    PubMed Central

    Ulker, Mustafa; Ulker, H. Esra; Zortuk, Mustafa; Bulbul, Mehmet; Tuncdemir, Ali Riza; Bilgin, M. Selim

    2009-01-01

    Objectives: The aim of the present study was to evaluate the cytotoxic effects of three different provisional restoration materials on fibroblasts. Two bis-acrylic based [Tempofit Duomix (Detax), Protemp 3 Garant (3M ESPE)] and one urethan dimethacrylate [Revotek LC (GC Corporation)] based provisional restoration materials used. Methods: Materials were prepared according to the manufacturers’ instructions in standard teflon disks (2×5 mm) and four samples were extracted in 7 ml of Basal Medium Eagle with 10% new born calf serum and 100 mg/ml penicillin/streptomycin for 24 hours. The L929 fibroblast cells were plated (25.000 cells/ml) in well plates, and maintained in a CO2 incubator at 37°C for 24h. After 24 hours, the incubation medium was replaced by the immersed medium in which the samples were stored and the L929 fibroblasts were incubated in contact with eluates for 24 hours at 37°C for 24h. The fibroblast cell viability was analyzed by measuring the mitochondrial activity with the methyltetrazolium test (MTT). Twelve well used for each specimen and experiment repeated for two times. The data was statistically analyzed by Mann-Whitney U tests. Results: The results showed that, Revotek LC and Protemp 3 Garant were not cytotoxic for fibroblast cells when compared to control group (P>.05). However, Tempofit duomix was cytotoxic for L929 fibroblasts when compared to control group and other tested materials (P<.05). Conclusions: Taking into consideration the limitations of an in vitro study, our study indicate that provisional restoration materials might have cytotoxic effects on fibroblasts and should be selected carefully for clinical applications. PMID:19421391

  11. Stimulatory effects of distinct members of the bone morphogenetic protein family on ligament fibroblasts

    PubMed Central

    Bobacz, K; Ullrich, R; Amoyo, L; Erlacher, L; Smolen, J S; Graninger, W B

    2006-01-01

    Objective To investigate effects of cartilage derived morphogenetic protein‐1 and ‐2 (CDMP‐1, CDMP‐2), bone morphogenetic protein (BMP)‐7 and BMP‐6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. Methods Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6. Expression of the indicated growth factors was assessed by RT‐PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT‐PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [35S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. Results CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [35S]sulphate incorporation. Toluidine blue staining showed cartilage‐specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. Conclusion CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP‐1, CDMP‐2, BMP‐7, and BMP‐6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair. PMID:15975973

  12. Transcriptomic study of high‑glucose effects on human skin fibroblast cells.

    PubMed

    Pang, Lingxia; Wang, Youpei; Zheng, Meiqin; Wang, Qing; Lin, Hong; Zhang, Liqing; Wu, Lingjian

    2016-03-01

    Skin ulcers are a common complication of diabetes mellitus (DM). Fibroblasts are located within the dermis of skin tissue and can be damaged by diabetes. However, the underlying mechanism of how DM affects fibroblasts remains elusive. To understand the effects of DM on fibroblasts, the current study mimicked DM by high‑glucose (HG) supplementation in the culture medium of human foreskin primary fibroblast cells, and the analysis of transcriptomic changes was conducted. RNA sequencing‑based transcriptome analysis identified that, upon HG stress, 463 genes were upregulated and 351 genes downregulated (>1.5‑fold changes; P<0.05). These altered genes were distributed into 20 different pathways. In addition, gene ontology (GO) analysis indicated that 31 GO terms were enriched. Among the pathways identified, nuclear factor κB (NF‑κB) pathway genes were highly expressed, and the addition of Bay11‑7082, a typical NF‑κB signaling inhibitor, blocked the previously observed alterations in plasminogen activator inhibitor 1 (PAI1), an inflammation marker and frizzled class receptor 8 (FZD8), a Wnt signaling gene, expression that resulted from HG stress. Furthermore, an inhibitor of Wnt signaling diminished the role of Bay11‑7082 in the regulation of PAI1 expression under HG conditions, suggesting that Wnt signaling may function downstream of the NF‑κB pathway to protect fibroblast cells from HG stress. To the best of our knowledge, the current study is the first analysis of transcriptomic responses under HG stress in human fibroblasts. The data provided here may aid the understanding of the molecular mechanisms by which fibroblast cells are damaged in the skin of patients with DM. PMID:26820167

  13. Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate.

    PubMed

    Djouadi, Fatima; Habarou, Florence; Le Bachelier, Carole; Ferdinandusse, Sacha; Schlemmer, Dimitri; Benoist, Jean François; Boutron, Audrey; Andresen, Brage S; Visser, Gepke; de Lonlay, Pascale; Olpin, Simon; Fukao, Toshiyuki; Yamaguchi, Seiji; Strauss, Arnold W; Wanders, Ronald J A; Bastin, Jean

    2016-01-01

    Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 μM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes. PMID:26109258

  14. Antiproliferative effect of methanolic extraction of tualang honey on human keloid fibroblasts

    PubMed Central

    2011-01-01

    Background Keloid is a type of scar which extends beyond the boundaries of the original wound. It can spread to the surrounding skin by invasion. The use of Tualang honey is a possible approach for keloid treatment. The objective of this study was to determine the antiproliferative effect of methanolic extraction of Tualang honey to primary human keloid fibroblasts and to identify the volatile compounds in methanol extraction of Tualang honey. Methods Crude Tualang honey was extracted with methanol and then dried using rota vapor to remove remaining methanol from honey. Normal and keloid fibroblasts were verified and treated with the extracted honey. Cell proliferation was tested with [3-(4,5-dimethylthiazol-2-yi)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay. Extraction of Tualang honey using methanol was carried out and the extracted samples were analysed using gas chromatography-mass spectrometry (GC-MS). The result was analysed using SPSS and tested with Kruskal-Wallis and Mann-Whitney tests. Results Methanolic extraction of honey has positive anti proliferative effect on keloid fibroblasts in a dose-dependent manner. The presence of fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid and octadecanoic acid may contribute to the anti-proliferative effect in keloid fibroblasts. Conclusions The methanolic honey extraction has an antiproliferative effect on keloid fibroblasts and a range of volatile compounds has been identified from Tualang honey. The antiproliferative effect of keloid fibroblasts towards Tualang honey may involve cell signaling pathway. Identifying other volatile compounds from different organic solvents should be carried out in future. PMID:21943200

  15. The effects of Sarconesiopsis magellanica larvae (Diptera: Calliphoridae) excretions and secretions on fibroblasts.

    PubMed

    Pinilla, Yudi T; Patarroyo, Manuel A; Velandia, Myriam L; Segura, Nidya A; Bello, Felio J

    2015-02-01

    Sarconesiopsis magellanica is a necrophagous blowfly which is relevant in both forensic and medical sciences. Previous studies regarding this species have led to understanding life-cycle, population and reproduction parameters, as well as identifying and characterising proteolytic enzymes derived from larval excretions and secretions (ES). As other studies have shown that ES proteolytic activity plays a significant role in wound healing and fibroblasts play a relevant role in granulation tissue formation during such healing, the present study was aimed at analysing the biological effect of S. magellanica larval ES on fibroblasts. ES were obtained from third-instar larvae and added to fibroblast cells at three concentrations (10, 5 and 1 μg/mL) to evaluate their behaviour. MTT assays were used for analysing cell proliferation and viability, whilst cell adhesion was measured by optical density with 10% SDS. Fibroblast migration and morphology was recorded by microscopic observation. ES did not affect fibroblast viability and induced an increase in cell proliferation; cell adhesion became reduced, whilst cell migration through extracellular matrix increased. ES also induced a decreased cell surface and morphological alterations. Changes in all the above-mentioned parameters were reduced when ES were incubated at 60 °C, probably due to protease denaturation. These results suggested that the proteases contained in S. magellanica larval ES contributed towards granulation tissue formation, increased cell migration and promoted cell proliferation. All these data support carrying out further experiments aimed at validating S. magellanica usefulness in larval therapy. PMID:25445745

  16. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide.

    PubMed

    Feng, Bing; Ma, Lai-ji; Yao, Jin-jing; Fang, Yun; Mei, Yan-ai; Wei, Shao-min

    2013-02-01

    Oat contains different components that possess antioxidant properties; no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level. Therefore, the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide (H(2)O(2)). Kjeldahl determination, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%, of which 97.43% are peptides with a molecular range from 438.56 to 1301.01 Da. Assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay for apoptosis showed that administration of H(2)O(2) in human dermal fibroblasts caused cell damage and apoptosis. Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H(2)O(2), but application oat peptides with H(2)O(2) at same time did not. Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H(2)O(2)-induced decrease of superoxide dismutase (SOD) and the inhibition of malondialdehyde (MDA). The results demonstrate that oat peptides possess antioxidant activity and are effective against H(2)O(2)-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level. Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury. PMID:23365008

  17. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide

    PubMed Central

    Feng, Bing; Ma, Lai-ji; Yao, Jin-jing; Fang, Yun; Mei, Yan-ai; Wei, Shao-min

    2013-01-01

    Oat contains different components that possess antioxidant properties; no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level. Therefore, the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide (H2O2). Kjeldahl determination, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%, of which 97.43% are peptides with a molecular range from 438.56 to 1 301.01 Da. Assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay for apoptosis showed that administration of H2O2 in human dermal fibroblasts caused cell damage and apoptosis. Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H2O2, but application oat peptides with H2O2 at same time did not. Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H2O2-induced decrease of superoxide dismutase (SOD) and the inhibition of malondialdehyde (MDA). The results demonstrate that oat peptides possess antioxidant activity and are effective against H2O2-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level. Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury. PMID:23365008

  18. p16(INK4A) represses the paracrine tumor-promoting effects of breast stromal fibroblasts.

    PubMed

    Al-Ansari, M M; Hendrayani, S F; Shehata, A I; Aboussekhra, A

    2013-05-01

    Cancer-associated fibroblasts (CAFs), the most abundant and probably the most active cellular component of breast cancer-associated stroma, promote carcinogenesis through paracrine effects; however, the molecular basis remains elusive. We have shown here that p16(INK4A) expression is reduced in 83% CAFs as compared with their normal adjacent counterparts cancer-free tissues isolated from the same patients. This decrease is mainly due to AUF1-dependent higher turnover of the CDKN2A mRNA in CAFs. Importantly, p16(INK4A) downregulation using specific siRNA activated breast fibroblasts and increased the expression/secretion levels of stromal cell-derived factor 1 (SDF-1) and matrix metalloproteinase (MMP)-2. Consequently, media conditioned with these cells stimulated the proliferation of epithelial cells. Furthermore, the migration/invasion of breast cancer cells was also enhanced in an SDF-1-dependent manner. This effect was mediated through inducing an epithelial-mesenchymal transition state. By contrast, increase in p16(INK4A) level through ectopic expression or AUF1 downregulation, reduced the secreted levels of SDF-1 and MMP-2 and suppressed the pro-carcinogenic effects of CAFs. In addition, p16(INK4A)-defective fibroblasts accelerated breast tumor xenograft formation and growth rate in mice. Importantly, tumors formed in the presence of p16(INK4A)-defective fibroblasts exhibited higher levels of active Akt, Cox-2, MMP-2 and MMP-9, showing their greater aggressiveness as compared with xenografts formed in the presence of p16(INK4A)-proficient fibroblasts. These results provide the first indication that p16(INK4A) downregulation in breast stromal fibroblasts is an important step toward their activation. PMID:22751126

  19. Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte

    PubMed Central

    Jeong, Dayeong; Han, Chungmin; Kang, Inhye; Park, Hyun Taek; Kim, Jiyoon; Ryu, Hayoung; Gho, Yong Song; Park, Jaesung

    2016-01-01

    The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells. PMID:26863621

  20. The effects of levofloxacin on rabbit fibroblast-like synoviocytes in vitro

    SciTech Connect

    Tan, Yang; Lu, Kaihang; Deng, Yu; Cao, Hong; Chen, Biao; Wang, Hui; Magdalou, Jacques; Chen, Liaobin

    2012-12-01

    It is widely accepted that tendon and cartilage are adversely affected with the toxic effects of quinolones. However, the effects of quinolones on synovium have not been deciphered completely. In this study, our main objective was to investigate the effects of levofloxacin, a typical quinolone antibiotic drug, on fibroblast-like synoviocytes (FLSs) in vitro. FLSs of rabbits were treated with levofloxacin at different concentrations (0, 14, 28, 56, 112 and 224 μM). The possible cytotoxic effects of levofloxacin on FLS were determined. Levofloxacin significantly reduced the cell viabilities, gene expression of hyaluronan synthase-2 (HAS-2), and the level of hyaluronan in FLSs. Moreover, levofloxacin-induced concentration-dependent increases of apoptosis and active caspase-3 were determined in this study. Ultrastructural damages of FLSs were observed by electron microscopy. The mRNA expression levels of matrix metalloproteinase (MMP)-3 and MMP-13 were increased in FLSs treated with levofloxacin. In addition, levofloxacin played a role in suppressing the expression of interleukin (IL)-1 and IL-6. Our data suggest that the cytotoxic effects of levofloxacin on FLS were shown to be able to affect cell viability and HA synthesis capacity. The potential mechanisms of the cytotoxic effects may be attributed to the apoptosis and increased expression of MMPs. -- Highlights: ► Levofloxacin decreases hyaluronic acid synthesis in fibroblast-like synoviocytes. ► Levofloxacin exerts pro-apoptosis effects on fibroblast-like synoviocytes. ► Levofloxacin increases gene expression of MMPs in fibroblast-like synoviocytes. ► Levofloxacin exerts anti-inflammatory effects on fibroblast-like synoviocytes.

  1. Paracrine anti-fibrotic effects of neonatal cells and living cell constructs on young and senescent human dermal fibroblasts.

    PubMed

    Pratsinis, Harris; Armatas, Andreas; Dimozi, Anastasia; Lefaki, Maria; Vassiliu, Pantelis; Kletsas, Dimitris

    2013-01-01

    Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs--in the form of a conditioned medium (CM)--on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha-smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress-induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC-derived CM was found to be more potent than fibroblast-derived CMs and, furthermore, to inhibit alpha-smooth muscle actin expression. In conclusion, these results may indicate anti-contractile and anti-fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor-derived fibroblasts. These activities seem to persist during senescence of the target cells. PMID:24581241

  2. Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

  3. Optimization of permeabilization process of yeast cells for catalase activity using response surface methodology

    PubMed Central

    Trawczyńska, Ilona; Wójcik, Marek

    2015-01-01

    Biotransformation processes accompanied by whole yeast cells as biocatalyst are a promising area of food industry. Among the chemical sanitizers currently used in food technology, hydrogen peroxide is a very effective microbicidal and bleaching agent. In this paper, permeabilization has been applied to Saccharomyces cerevisiae yeast cells aiming at increased intracellular catalase activity for decomposed H2O2. Ethanol, which is non-toxic, biodegradable and easily available, has been used as permeabilization factor. Response surface methodology (RSM) has been applied in determining the influence of different parameters on permeabilization process. The aim of the study was to find such values of the process parameters that would yield maximum activity of catalase during decomposition of hydrogen peroxide. The optimum operating conditions for permeabilization process obtained by RSM were as follows: 53% (v/v) of ethanol concentration, temperature of 14.8 °C and treatment time of 40 min. After permeabilization, the activity of catalase increased ca. 40 times and its maximum value equalled to 4711 U/g. PMID:26019618

  4. Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology

    PubMed Central

    de Faria, Janaína T.; Rocha, Pollyana F.; Converti, Attilio; Passos, Flávia M.L.; Minim, Luis A.; Sampaio, Fábio C.

    2013-01-01

    The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L−1 oNP min−1 g−1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry. PMID:24688494

  5. NAADP-sensitive Ca2+ stores in permeabilized rat hepatocytes.

    PubMed

    Bychkova, S V; Chorna, T I

    2014-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide that is potent to release calcium from intracellular stores in different cell types. NAADP was shown to target specific type of intracellular store namely endolysosomal system or acidic store. Despite intense studies, its effect on endoplasmatic reticulum (ER) still remains to be elucidated. The main aim of our work was to investigate NAADP-sensitive store in permeabilized rat hepatocytes monitoring the level of Ca2+ inside intracellular organelles using chlorotetracycline (CTC). We have shown that NAADP triggered changes of stored Ca2+ in rat hepatocytes are dependent on concentration of EGTA-Ca2+-buffer in cell incubation medium, i.e. the higher is the EGTA concentration in incubation medium the smaller or absent is the effect of NAADP. Besides, the effect of NAADP was more pronounced upon cells pretreatment with the inhibitory concentration of ryanodine (100 μM). This might suggest that the effect of NAADP is dependent on ER luminal calcium. We have also found that NAADP-evoked Ca2+ release in permeabilized hepatocytes is sensitive to nigericin, bafilomycin A and thapsigargin. Additionally, NAADP triggered changes in stored Ca2+ were completely abolished by NED-19 as antagonist of NAADP. PMID:25816589

  6. Effects of Mechanical Stretching on the Morphology and Cytoskeleton of Vaginal Fibroblasts from Women with Pelvic Organ Prolapse

    PubMed Central

    Wang, Sumei; Zhang, Zhenyu; Lü, Dongyuan; Xu, Qiuxiang

    2015-01-01

    Mechanical load and postmenopausal hypoestrogen are risk factors for pelvic organ prolapse (POP). In this study, we applied a 0.1-Hz uniaxial cyclic mechanical stretching (CS) with 10% elongation and 10−8 M 17-β-estradiol to vaginal fibroblasts isolated from postmenopausal women with or without POP to investigate the effects of CS and estrogen on cell morphology and cytoskeletons of normal and POP fibroblasts. Under static culture condition, POP fibroblasts exhibited lower cell circularity and higher relative fluorescence intensities (RFIs) of F-actin, α-tubulin and vimentin. When cultured with CS, all fibroblasts grew perpendicular to the force and exhibited a decreased cell projection area, cell circularity and increased cell length/width ratio; normal fibroblasts exhibited increased RFIs of all three types of cytoskeleton, and POP fibroblasts exhibited a decreased RFI of F-actin and no significant differences of α-tubulin and vimentin. After being cultured with 17-β-estradiol and CS, normal fibroblasts no longer exhibited significant changes in the cell projection area and the RFIs of F-actin and α-tubulin; POP fibroblasts exhibited no significant changes in cell circularity, length/width ratio and F-actin even with the increased RFIs of α-tubulin and vimentin. These findings suggest that POP fibroblasts have greater sensitivity to and lower tolerance for mechanical stretching, and estrogen can improve the prognosis. PMID:25923074

  7. Effects of mechanical stretching on the morphology and cytoskeleton of vaginal fibroblasts from women with pelvic organ prolapse.

    PubMed

    Wang, Sumei; Zhang, Zhenyu; Lü, Dongyuan; Xu, Qiuxiang

    2015-01-01

    Mechanical load and postmenopausal hypoestrogen are risk factors for pelvic organ prolapse (POP). In this study, we applied a 0.1-Hz uniaxial cyclic mechanical stretching (CS) with 10% elongation and 10⁻⁸ M 17-β-estradiol to vaginal fibroblasts isolated from postmenopausal women with or without POP to investigate the effects of CS and estrogen on cell morphology and cytoskeletons of normal and POP fibroblasts. Under static culture condition, POP fibroblasts exhibited lower cell circularity and higher relative fluorescence intensities (RFIs) of F-actin, α-tubulin and vimentin. When cultured with CS, all fibroblasts grew perpendicular to the force and exhibited a decreased cell projection area, cell circularity and increased cell length/width ratio; normal fibroblasts exhibited increased RFIs of all three types of cytoskeleton, and POP fibroblasts exhibited a decreased RFI of F-actin and no significant differences of α-tubulin and vimentin. After being cultured with 17-β-estradiol and CS, normal fibroblasts no longer exhibited significant changes in the cell projection area and the RFIs of F-actin and α-tubulin; POP fibroblasts exhibited no significant changes in cell circularity, length/width ratio and F-actin even with the increased RFIs of α-tubulin and vimentin. These findings suggest that POP fibroblasts have greater sensitivity to and lower tolerance for mechanical stretching, and estrogen can improve the prognosis. PMID:25923074

  8. Plasma Membrane Permeabilization by Trains of Ultrashort Electric Pulses

    PubMed Central

    Ibey, Bennett L.; Mixon, Dustin G.; Payne, Jason A.; Bowman, Angela; Sickendick, Karl; Wilmink, Gerald J.; Roach, W. Patrick; Pakhomov, Andrei G.

    2010-01-01

    Ultrashort electric pulses (USEP) cause long-lasting increase of cell membrane electrical conductance, and that a single USEP increased cell membrane electrical conductance proportionally to the absorbed dose (AD) with a threshold of about 10 mJ/g. The present study extends quantification of the membrane permeabilization effect to multiple USEP and employed a more accurate protocol that identified USEP effect as the difference between post- and pre-exposure conductance values (Δg) in individual cells. We showed that Δg can be increased by either increasing the number of pulses at a constant E-field, or by increasing the E-field at a constant number of pulses. For 60-ns pulses, an E-field threshold of 6 kV/cm for a single pulse was lowered to less than 1.7 kV/cm by applying 100-pulse or longer trains. However, the reduction of the E-field threshold was only achieved at the expense of a higher AD compared to a single pulse exposure. Furthermore, the effect of multiple pulses was not fully determined by AD, suggesting that cells permeabilized by the first pulse(s) in the train become less vulnerable to subsequent pulses. This explanation was corroborated by a model that treated multiple-pulse exposures as a series of single-pulse exposures and assumed an exponential decline of cell susceptibility to USEP as Δg increased after each pulse during the course of the train. PMID:20171148

  9. Distinctive Effects of Cytochalasin B in Chick Primary Myoblasts and Fibroblasts

    PubMed Central

    de Andrade, Ivone Rosa; Costa, Manoel Luis; Mermelstein, Claudia

    2016-01-01

    Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3–8 μm in width, and >200 μm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells. PMID:27119825

  10. The effectiveness of potent dental adhesives on the viability of LPS challenged human gingival fibroblasts.

    PubMed

    Garner, Angelia D; Tucci, Michelle A; Benghuzzi, Hamed A

    2014-01-01

    Dental adhesives are necessary for the retention of specific dental restorations utilized to repair the anatomy of the tooth after dental decay is removed. Adhesives come into contact with healthy and diseased periodontal tissues. Porphyromonas gingivalis is a gram negative bacterial pathogen, and lipopolysaccharide (LPS-PG) is an endotoxin found in gingival connective tissues of patients who suffer from periodontal disease. The presence of the endotoxin causes inflammation. This study aims to evaluate the effectiveness of potent dental adhesives when human gingival fibroblasts are challenged with LPS-PG. The fibroblasts were exposed to the dental adhesives polymethly methacrylate (PMMA), OptiBond®, and Prime & Bond® which were purchased from Patterson Dental, a national dental materials supplier. The human gingival fibroblasts (HGF-1, ATCC® CRL-2014™) were purchased from American Type Culture Collection (ATCC). The porphyromonas gingival lipopolysaccharide (LPS-PG) was purchased from Fisher Scientific (Pittsburg, PA). No significant differences in metabolic behavior was detected among the groups (p<0.132). While the glutathione assay determined that there was not any significant increase in oxidative stress levels; the lactate dehydrogenase assay identified significant cellular damage in the group exposed to combinations of the Prime & Bond® adhesives and LPS-PG at 48 hour intervals (p<0.003). No significant changes were noted in cellular morphology at any phases, and all cells demonstrated typical fibroblast spindle shape. PMID:25405402

  11. Roles of charged particles and reactive species on cell membrane permeabilization induced by atmospheric-pressure plasma irradiation

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Hokari, Yutaro; Tominami, Kanako; Mokudai, Takayuki; Kanetaka, Hiroyasu; Kaneko, Toshiro

    2016-07-01

    As factors that influence cell membrane permeabilization during direct and indirect atmospheric-pressure plasma irradiation, charged particle influx, superoxide anion radicals (O2 ‑•), and hydrogen peroxide (H2O2) in plasma-irradiated solution were evaluated. These are the three strong candidate factors and might multiply contribute to cell membrane permeabilization. In particular, a shorter plasma diffusion distance leads to the enhancement of the direct effects such as charged particle influx and further increase cell membrane permeability. In addition, O2 ‑• dissipates over time (a life span of the order of minutes) in plasma-irradiated water, and the deactivation of a plasma-irradiated solution in term of cell membrane permeabilization occurs in a life span of the same order. These results could promote the understanding of the mechanism of plasma-induced cell membrane permeabilization.

  12. Effect of Lipopolysaccharide on Glucocorticoid Receptor Function in Control Nasal Mucosa Fibroblasts and in Fibroblasts from Patients with Chronic Rhinosinusitis with Nasal Polyps and Asthma

    PubMed Central

    Fernández-Bertolín, Laura; Mullol, Joaquim; Fuentes-Prado, Mireya; Roca-Ferrer, Jordi; Alobid, Isam; Picado, César; Pujols, Laura

    2015-01-01

    Background Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment. Objective We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma. Methods NP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing. Results Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/β MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone’s capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRβ expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone’s capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone’s capacity to suppress FBS-induced CXCL8 release. Conclusion The bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the

  13. Porphyrin–phospholipid liposomes permeabilized by near-infrared light

    PubMed Central

    Carter, Kevin A.; Shao, Shuai; Hoopes, Matthew I.; Luo, Dandan; Ahsan, Bilal; Grigoryants, Vladimir M.; Song, Wentao; Huang, Haoyuan; Zhang, Guojian; Pandey, Ravindra K.; Geng, Jumin; Pfeifer, Blaine A.; Scholes, Charles P.; Ortega, Joaquin; Karttunen, Mikko; Lovell, Jonathan F.

    2014-01-01

    The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin–phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin–phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin–phospholipid doping, irradiation intensity or irradiation duration. Porphyrin–phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy. PMID:24699423

  14. Porphyrin-phospholipid liposomes permeabilized by near-infrared light.

    PubMed

    Carter, Kevin A; Shao, Shuai; Hoopes, Matthew I; Luo, Dandan; Ahsan, Bilal; Grigoryants, Vladimir M; Song, Wentao; Huang, Haoyuan; Zhang, Guojian; Pandey, Ravindra K; Geng, Jumin; Pfeifer, Blaine A; Scholes, Charles P; Ortega, Joaquin; Karttunen, Mikko; Lovell, Jonathan F

    2014-01-01

    The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin-phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin-phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin-phospholipid doping, irradiation intensity or irradiation duration. Porphyrin-phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy. PMID:24699423

  15. Antiviral effects of human fibroblast interferon from Escherichia coli against encephalomyocarditis virus infection of squirrel monkeys.

    PubMed

    Weck, P K; Harkins, R N; Stebbing, N

    1983-02-01

    Recombinant DNA methodology has allowed the production of human fibroblast interferon (IFN-beta) from Escherichia coli and this material, in highly purified form, has been shown to reduce viraemia and mortality in encephalomyocarditis (EMC) virus-infected squirrel monkeys. These effects are dose related: six treatments over 4 days at 10(6) U/kg and 3 x 10(3) U/kg have comparable efficacy, whereas treatments at 10(3) U/kg are ineffective. The recombinant DNA-derived IFN-beta appears to be as effective as natural fibroblast cell-derived IFN-beta and both materials are effective by the intramuscular or intravenous routes. Thus, even though previous studies have shown that low circulating concentrations of IFN-beta are observed after intramuscular injections, the present data indicate that this slow release from the muscle can still confer protection. PMID:6300291

  16. Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

    PubMed

    Lee, Geum-Young; Park, Kang-Gyun; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2016-03-01

    Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml. PMID:26507878

  17. Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells.

    PubMed

    Clegg, J S; Jackson, S A

    1990-05-01

    L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization. PMID:2109584

  18. Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model.

    PubMed

    Okazaki, Mutsumi; Yoshimura, Kotaro; Suzuki, Yasutoshi; Harii, Kiyonori

    2003-09-01

    The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial

  19. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells.

    PubMed

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients' outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCMTGF, FCMPDGF) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCMB). FCMTGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCMTGF≫FCMPDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCMTGF>FCMPDGF) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. PMID:24394543

  20. The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

    PubMed

    Futakuchi, Akiko; Inoue, Toshihiro; Fujimoto, Tomokazu; Inoue-Mochita, Miyuki; Kawai, Motofumi; Tanihara, Hidenobu

    2016-08-01

    The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-β2, followed by incubation for 48 h. TGF-β2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-β2-induced fibronectin production and collagen gel contraction. TGF-β2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery. PMID:27394186

  1. Effects of growth factors on the proliferation of human keratinocytes and fibroblasts in vitro.

    PubMed

    Kim, D S; Korting, H C; Schäfer-Korting, M

    1998-01-01

    Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development. The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated. The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of [3H]-thymidine. Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect. In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent. The drug already proved to be effective at very low concentrations (0.1 ng/ml). Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM). TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml. Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures. IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M. EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production. Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures. In fibroblasts, GDF-5 induced very weak antiproliferative effects. Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF. There was, however, a contradiction between TGF-beta 1 results on fibroblasts. Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed

  2. Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

    PubMed Central

    Saw, Valerie P.J.; Offiah, Ifeoma; Dart, Robin J.; Galatowicz, Grazyna; Dart, John K.G.; Daniels, Julie T.; Calder, Virginia L.

    2009-01-01

    Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts. PMID:19910508

  3. Effect of Microalgal Extracts of Tetraselmis suecica against UVB-Induced Photoaging in Human Skin Fibroblasts.

    PubMed

    Jo, Wol Soon; Yang, Kwang Mo; Park, Hee Sung; Kim, Gi Yong; Nam, Byung Hyouk; Jeong, Min Ho; Choi, Yoo Jin

    2012-12-01

    Exposure of cells to ultraviolet B (UVB) radiation can induce production of free radicals and reactive oxygen species (ROS), which damage cellular components. In addition, these agents can stimulate the expression of matrix metalloproteinase (MMP) and decrease collagen synthesis in human skin cells. In this study, we examined the anti-photoaging effects of extracts of Tetraselmis suecica (W-TS). W-TS showed the strongest scavenging activity against 2,2-difenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. We observed that the levels of both intracellular ROS and lipid peroxidation significantly increased in UVB-irradiated human skin fibroblast cells. Furthermore, the activities of enzymatic antioxidants (e.g., superoxide dismutase) and the levels of non-enzymatic antioxidants (e.g., glutathione) significantly decreased in cells. However, W-TS pretreatment, at the maximum tested concentration, significantly decreased intracellular ROS and malondialdehyde (MDA) levels, and increased superoxide dismutase and glutathione levels in the cells. At this same concentration, W-TS did not show cytotoxicity. Type 1 procollagen and MMP-1 released were quantified using RT-PCR techniques. The results showed that W-TS protected type 1 procollagen against UVBinduced depletion in fibroblast cells in a dose-dependent manner via inhibition of UVB-induced MMP-1. Taken together, the results of the study suggest that W-TS effectively inhibits UVB-induced photoaging in skin fibroblasts by its strong anti-oxidant ability. PMID:24278616

  4. Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress.

    PubMed

    Richard, M J; Guiraud, P; Leccia, M T; Beani, J C; Favier, A

    1993-01-01

    In purified system zinc has been shown to have an antioxidant role. Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined. Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150 microM as Zinc chlorur ZnCl2). In comparison, cell controls were grown in standard culture media (6.5 microM Zn). The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330 +/- 120 ng/mg protein in 150-microM Zn-treated cells versus 331 +/- 21 ng/mg protein in control cells). The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased. The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 mumol/g protein in 150 microM Zn-treated cells versus 1.59 mumol/g protein in controls). At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities. Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage. However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H2O2), an increased toxicity in Zn-supplemented cells was observed. When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium. If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes. PMID:7688532

  5. Effect of static magnetic fields and phloretin on antioxidant defense system of human fibroblasts.

    PubMed

    Pawłowska-Góral, Katarzyna; Kimsa-Dudek, Magdalena; Synowiec-Wojtarowicz, Agnieszka; Orchel, Joanna; Glinka, Marek; Gawron, Stanisław

    2016-08-01

    The available evidence from in vitro and in vivo studies is deemed not sufficient to draw conclusions about the potential health effects of static magnetic field (SMF) exposure. Therefore, the aim of the present study was to determine the influence of static magnetic fields and phloretin on the redox homeostasis of human dermal fibroblasts. Control fibroblasts and fibroblasts treated with phloretin were subjected to the influence of static magnetic fields. Three chambers with static magnetic fields of different intensities (0.4, 0.55, and 0.7 T) were used in the study. Quantification of superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPX1), microsomal glutathione S-transferase 1 (MGST1), glutathione reductase (GSR), and catalase (CAT) messenger RNAs (mRNAs) was performed by means of real-time reverse transcription PCR (QRT-PCR) technique. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were measured using a commercially available kit. No significant differences were found in SOD1, SOD2, GPX1, MGST1, GSR, and CAT mRNA levels among the studied groups in comparison to the control culture without phloretin and without the magnet. There were also no changes in SOD, GPx, and CAT activities. In conclusion, our study indicated that static magnetic fields generated by permanent magnets do not exert a negative influence on the oxidative status of human dermal fibroblasts. Based on these studies, it may also be concluded that phloretin does not increase its antioxidant properties under the influence of static magnetic fields. However, SMF-induced modifications at the cellular and molecular level require further clarification. PMID:27080405

  6. Substrate effect modulates adhesion and proliferation of fibroblast on graphene layer.

    PubMed

    Lin, Feng; Du, Feng; Huang, Jianyong; Chau, Alicia; Zhou, Yongsheng; Duan, Huiling; Wang, Jianxiang; Xiong, Chunyang

    2016-10-01

    Graphene is an emerging candidate for biomedical applications, including biosensor, drug delivery and scaffold biomaterials. Cellular functions and behaviors on different graphene-coated substrates, however, still remain elusive to a great extent. This paper explored the functional responses of cells such as adhesion and proliferation, to different kinds of substrates including coverslips, silicone, polydimethylsiloxane (PDMS) with different curing ratios, PDMS treated with oxygen plasma, and their counterparts coated with single layer graphene (SLG). Specifically, adherent cell number, spreading area and cytoskeleton configuration were exploited to characterize cell-substrate adhesion ability, while MTT assay was employed to test the proliferation capability of fibroblasts. Experimental outcome demonstrated graphene coating had excellent cytocompatibility, which could lead to an increase in early adhesion, spreading, proliferation, and remodeling of cytoskeletons of fibroblast cells. Notably, it was found that the underlying substrate effect, e.g., stiffness of substrate materials, could essentially regulate the adhesion and proliferation of cells cultured on graphene. The stiffer the substrates were, the stronger the abilities of adhesion and proliferation of fibroblasts were. This study not only deepens our understanding of substrate-modulated interfacial interactions between live cells and graphene, but also provides a valuable guidance for the design and application of graphene-based biomaterials in biomedical engineering. PMID:27451366

  7. Effect of Collagen Nanotopography on Keloid Fibroblast Proliferation and Matrix Synthesis: Implications for Dermal Wound Healing

    PubMed Central

    Muthusubramaniam, Lalitha; Zaitseva, Tatiana; Paukshto, Michael; Martin, George

    2014-01-01

    Keloids are locally exuberant dermal scars characterized by excessive fibroblast proliferation and matrix accumulation. Although treatment strategies include surgical removal and intralesional steroid injections, an effective regimen is yet to be established due to a high rate of recurrence. The regressing center and growing margin of the keloid have different collagen architecture and also differ in the rate of proliferation. To investigate whether proliferation is responsive to collagen topography, keloid, scar, and dermal fibroblasts were cultured on nanopatterned scaffolds varying in collagen fibril diameter and alignment-small and large diameter, aligned and random fibrils, and compared to cells grown on flat collagen-coated substrates, respectively. Cell morphology, proliferation, and expression of six genes related to proliferation (cyclin D1), phenotype (α-smooth muscle actin), and matrix synthesis (collagens I and III, and matrix metalloproteinase-1 and -2) were measured to evaluate cell response. Fibril alignment was shown to reduce proliferation and matrix synthesis in all three types of fibroblasts. Further, keloid cells were found to be most responsive to nanotopography. PMID:24724556

  8. Modeled Osteopathic Manipulative Treatments: A Review of Their in Vitro Effects on Fibroblast Tissue Preparations.

    PubMed

    Zein-Hammoud, Manal; Standley, Paul R

    2015-08-01

    A key osteopathic tenet involves the body's ability to self-heal. Osteopathic manipulative treatment (OMT) has been evolved to improve this healing capacity. The authors' in vitro work has focused on modeling 2 common OMT modalities: myofascial release (MFR) and counterstrain. Their studies have evaluated the effects of these modalities on wound healing, cytokine secretion, and muscle repair. The key components of the host response to mechanical forces are fibroblasts, which are the main fascial cells that respond to different types of strain by secreting anti-inflammatory chemicals and growth factors, thus improving wound healing and muscle repair processes. The purpose of this review is to discuss the cellular and molecular mechanisms by which MFR and other OMT modalities work, in particular, the role of strained fibroblasts in inflammation, wound healing, and muscle repair and regeneration. Changing MFR parameters, such as magnitude, duration, direction, and frequency of strain, might uniquely affect the physiologic response of fibroblasts, muscle contraction, and wound healing. If such results are clinically translatable, the mechanisms underlying the clinical outcomes of OMT modalities will be better understood, and these treatments will be more widely accepted as evidence-based, first-line therapies. PMID:26214822

  9. Effects of UVB Radiation on the Physicochemical Properties of Fibroblasts and Keratinocytes.

    PubMed

    Dobrzyńska, Izabela; Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew A

    2016-06-01

    The skin is the largest human organ, providing the first line of defense to protect the body from physical and environmental effects. The aim of this study was to determine the influence of short-wave ultraviolet (UVB) radiation on the membrane electrical properties, phospholipid content, and lipid peroxidation levels of fibroblasts and keratinocytes. Changes in cell function may affect the basal electrical surface properties of cell membranes. These changes can be detected using electrokinetic measurements. In this study, the surface charge densities of fibroblasts and keratinocytes were measured as a function of pH. A four-component equilibrium model was used to describe the interaction between the ions in solution and on cell membrane surfaces. Agreement was found between the experimental and theoretical charge variation curves of leukemia cells from pH 2.5 to pH 9. Phospholipid composition was determined qualitatively and quantitatively by HPLC, and lipid peroxidation was estimated by measuring the level of malondialdehyde. The acidic functional group concentrations and average association constants with hydroxyl ions were higher, and the average association constants with hydrogen ions were smaller in UVB-treated skin cell membranes compared to those in untreated cells. Moreover, our results showed that UVB radiation is associated with increased levels of phospholipids and lipid peroxidation products in fibroblasts and keratinocytes. PMID:26809654

  10. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    SciTech Connect

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  11. Light-triggered liposomal release: membrane permeabilization by photodynamic action.

    PubMed

    Pashkovskaya, Alina; Kotova, Elena; Zorlu, Yunus; Dumoulin, Fabienne; Ahsen, Vefa; Agapov, Igor; Antonenko, Yuri

    2010-04-20

    Photosensitized damage to liposome membranes was studied by using different dye-leakage assays based on fluorescence dequenching of a series of dyes upon their release from liposomes. Irradiation of liposomes with red light in the presence of a photosensitizer, trisulfonated aluminum phthalocyanine (AlPcS(3)), resulted in the pronounced leakage of carboxyfluorescein, but rather weak leakage of sulforhodamine B and almost negligible leakage of calcein from the corresponding dye-loaded liposomes. The same series of selectivity of liposome leakage was obtained with chlorin e6 that appeared to be more potent than AlPcS(3) in bringing about the photosensitized liposome leakage. Electrically neutral zinc phthalocyanine tetrasubstituted with a glycerol moiety (ZnPcGlyc(4)) was less effective than negatively charged AlPcS(3) in provoking the light-induced liposome permeabilization. On the contrary, both ZnPcGlyc(4) and AlPcS(3) were much more effective than chlorin e6 in sensitizing gramicidin channel inactivation in planar bilayer lipid membranes, thus showing that relative photodynamic efficacy of sensitizers can differ substantially for damaging different membrane targets. The photosensitized liposome permeabilization was apparently associated with oxidation of lipid double bonds by singlet oxygen as evidenced by the mandatory presence of unsaturated lipids in the membrane composition for the photosensitized liposome leakage to occur and the sensitivity of the latter to sodium azide. The fluorescence correlation spectroscopy measurements revealed marked permeability of photodynamically induced pores in liposome membranes for such photosensitizer as AlPcS(3). PMID:20000430

  12. Permeabilization and fusion of uncharged lipid vesicles induced by the HIV-1 fusion peptide adopting an extended conformation: dose and sequence effects.

    PubMed Central

    Pereira, F B; Goñi, F M; Muga, A; Nieva, J L

    1997-01-01

    fusion peptide what actively destabilizes cholesterol-containing, electrically neutral membranes. Moreover, membrane destabilization is modulated by the amino acid sequence in the extended structure. The effect displayed by the aforementioned V2 --> E substitution suggests that the fusion process described here could be reflecting a physiologically relevant phenomenon. Images FIGURE 3 PMID:9336193

  13. Protective effect of resveratrol against caspase 3 activation in primary mouse fibroblasts

    PubMed Central

    Ulakcsai, Zsófia; Bagaméry, Fruzsina; Vincze, István; Szökő, Éva; Tábi, Tamás

    2015-01-01

    Aim To study the effect of resveratrol on survival and caspase 3 activation in non-transformed cells after serum deprivation. Methods Apoptosis was induced by serum deprivation in primary mouse embryonic fibroblasts. Caspase 3 activation and lactate dehydrogenase release were assayed as cell viability measure by using their fluorogenic substrates. The involvement of PI3K, ERK, JNK, p38, and SIRT1 signaling pathways was also examined. Results Serum deprivation of primary fibroblasts induced significant activation of caspase 3 within 3 hours and reduced cell viability after 24 hours. Resveratrol dose-dependently prevented caspase activation and improved cell viability with 50% inhibitory concentration (IC50) = 66.3 ± 13.81 µM. It also reduced the already up-regulated caspase 3 activity when it was added to the cell culture medium after 3 hour serum deprivation, suggesting its rescue effect. Among the major signaling pathways, p38 kinase was critical for the protective effect of resveratrol which was abolished completely in the presence of p38 inhibitor. Conclusion Resveratrol showed protective effect against cell death in a rather high dose. Involvement of p38 kinase in this effect suggests the role of mild stress in its cytoprotective action. Furthermore due to its rescue effect, resveratrol may be used not only for prevention, but also treatment of age-related degenerative diseases, but in the higher dose than consumed in conventional diet. PMID:25891866

  14. The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts.

    PubMed

    Savage, K; Siebert, E; Swann, D

    1987-07-01

    The effect of platelet-derived growth factor (PDGF) on cell division and glycosaminoglycan (GAG) synthesis by fibroblasts isolated from skin and scar was measured. We found that PDGF stimulates cell division more efficiently in normal skin fibroblasts than in scar fibroblasts and decreases GAG synthesis in skin and scar fibroblasts. Using a 4-h pulse label with [3H]thymidine ([3H]Thd) following a 20-h incubation of confluent monolayer cultures with 0-5 units PDGF/ml Dulbecco's modified Eagle's medium, we found a concentration-dependent increase in [3H]Thd incorporation. After incubation of fibroblasts with [3H]glucosamine and 35SO4 in the presence or absence of PDGF, labeled constituents were isolated from the extracellular, pericellular, and cellular fractions by pronase digestion and column chromatography on Sepharose CL4B or DEAE-cellulose and analyzed by cellulose acetate electrophoresis. The presence of PDGF decreased the total amount of 35S incorporated into macromolecules by skin and scar fibroblasts and resulted in an altered distribution of labeled GAGs. Dermal fibroblasts exposed to PDGF for 24 h incorporated a greater percentage of radiolabeled 35S into dermatan sulfate prime (DS') and less into dermatan sulfate (DS) in the extracellular fractions and a greater percentage of 35S into heparan sulfate (HS) in the pericellular fractions than did parallel cultures grown in the absence of PDGF. It is thought than PDGF may have an effect on scar formation by increasing the fibroblast population in the wound tissue and by affecting the total amount and types of matrix components synthesized. PMID:3598205

  15. Effects of ascorbate and B-aminopropionitrile on tendon fibroblast migration in vitro

    SciTech Connect

    Nelson, J.M.; Cohen, I.K.; Diegelmann, R.F.

    1986-03-01

    Ascorbate (Asc) stimulates collagen synthesis and hydroxylation whereas beta-aminopropionitrile (BAPN) inhibits collagen cross-link formation. In this study, an in vitro model employing isolated chicken tendon biopsies (2 mm in dia.) in a fibrin clot has been used to examine the effect of Asc and BAPN on tendon fibroblast migration and proliferation. After 5 days in culture with either Asc (0.1 mM), or BAPN (0.5, 1.0, 2.0 mM), cell migration was measured using an automatic planimeter and cell proliferation was quantitated by /sup 125/IUDR incorporation into DNA. In cultures treated with Asc alone, cell migration was enhanced by approximately 33% compared to controls without ascorbate (5.9 mm/sup 2/ vs. 4.4 mm/sup 2/; p < 0.05) with no significant effect on cell proliferation. In contrast, cultures incubated with increasing concentrations of BAPN (0.5 - 2.0 mM) displayed a dose-dependent decrease (up to 9.6-fold at 2 mM) in fibroblast migration into the clot. The inhibitory effect of BAPN on cell migration was not due to a corresponding inhibition of fibroblast proliferation. These observations suggest that Asc enhanced collagen formation and thus allowed greater cell migration into the fibrin matrix. In contrast, in the absence of a mature, cross-linked collagen matrix following exposure to BAPN, cell migration was sub-optimal. These in vitro studies support the hypothesis that modulation of the collagen matrix may be a useful means of regulating tissue repair in vivo.

  16. Modulatory Effects of Connexin-43 Expression on Gap Junction Intercellular Communications with Mast Cells and Fibroblasts

    PubMed Central

    Pistorio, Ashey L.; Ehrlich, H. Paul

    2011-01-01

    The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junctional intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin in RMC-1 cells. Fibroblasts express other connexins that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited α SMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis. PMID:21328609

  17. Delivery of optical contrast agents using Triton-X100, part 1: reversible permeabilization of live cells for intracellular labeling

    NASA Astrophysics Data System (ADS)

    van de Ven, Anne L.; Adler-Storthz, Karen; Richards-Kortum, Rebecca

    2009-03-01

    Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility of Triton-X100-mediated permeabilization in live mammalian cells. We report a series of studies to characterize macromolecule delivery in cells following Triton-X100 treatment. Using this approach, we demonstrate that molecules ranging from 1 to 150 kDa in molecular weight can be reproducibly delivered into live cells by controlling the moles of Triton-X100 relative to the number of cells to be treated. When Triton-X100 is administered at or near the minimum effective concentration, cell permeabilization is generally reversed within 24 h, and treated cells continue to proliferate and show metabolic activity during the restoration of membrane integrity. We conclude that Triton-X100 is a promising permeabilization agent for efficient and reproducible delivery of optical contrast agents into live mammalian cells.

  18. Effects of dexamethasone on human synovial fibroblast-like cells, from osteoarthritic joints, in culture

    SciTech Connect

    Vento, R.; Torregrossa, M.V.; Giuliano, M.; Grecomoro, G.; Piccione, F. )

    1990-01-01

    The effect of Dexamethasone (DEX) on cell division and macromolecular synthesis was investigated in a line (Mc Coy cells, A 9) of synovial fibroblast-like cells derived from human osteoarthritic joints. DEX markedly reduced the proliferation of Mc Coy cells in a time and dose-dependent manner. The maximal inhibition was found at 500 nM DEX 24 h after incubation and was accompanied by the appearance of giant macrophage-like cells. After DEX treatment cells showed increased content of DNA, proteins and RNA together with the reduction of ({sup 3}H)-thymidine incorporation into the TCA-precipitable fraction.

  19. Effect of Nd:YAG collagen synthesis by human pulp fibroblasts

    NASA Astrophysics Data System (ADS)

    Barkhordar, Rahmat A.; Ghani, Qaiser P.; Enriquez, Belma A.; Hussain, M. Zamir

    1996-04-01

    The effect of Nd:YAG laser on tissue is dependant on treatment power, times and frequencies. In order to determine the biologic effect Nd:YAG laser radiation on dental pulp tissue, human dental pulp fibroblasts were obtained from extracted human teeth, and grown in DME containing 10% FBS and used as passage 6 - 7. Confluent cells were exposed to defocused laser energy (1 W, 10 Hz) using a pulsed (120 microsecond(s) ) fiberoptic delivered (320 micrometers fiber) Nd:YAG for 1 - 5 minutes. The cells were labeled with 10 (mu) Ci/ml of 3H proline for 16 hours. Untreated cultures were used as controls. Collagen synthesis was expressed as DPM X 10-3 mg/protein. The purpose of these measurements was to ascertain whether these radiation levels may induced a reparative response resulting in enhanced collagen synthesis. The results showed that at 1 - 5 minutes there was not significant effect. The tested lasing doses apparently did not cause any injurious effect to the fibroblasts, hence no increase in collagen synthesis.

  20. Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

    PubMed Central

    Ramirez, Allan M.; Wongtrakool, Cherry; Welch, Teresa; Steinmeyer, Andreas; Zügel, Ulrich; Roman, Jesse

    2010-01-01

    The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDR) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element – reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)2D3, showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)2D3 on fibroblasts treated with transforming growth factor β1 (TGFβ1), considered a driver of many fibrotic disorders, we found that 1,25(OH)2D3 inhibited TGFβ1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)2D3 also inhibited TGFβ1 stimulation of α-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFβ1-treated fibroblasts. Finally, we examined how 1,25(OH)2D3 affects epithelial-mesenchymal transformation of lung epithelial cells upon exposure to TGFβ1. We showed that the TGFβ1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)2D3. These observations suggest that under TGFβ1 stimulation, 1,25(OH)2D3 inhibits the profibrotic phenotype of lung fibroblasts and epithelial cells. PMID:19931390

  1. Effect of static magnetic fields on osteoblasts and fibroblasts in vitro.

    PubMed

    McDonald, F

    1993-01-01

    In vitro assays were made of the effect of a static magnetic field of a neodymium magnet on cellular behavior. The cell turnover rate was examined by the incorporation of radioactive thymidine, and anabolic processes were measured by the incorporation of radioactive proline. Cell cultures of fibroblast- and osteoblast-like cells of the neonatal rat calvarium were assayed to determine uptakes of radioactive thymidine and proline; these assays were performed in conjunction with examination of an explant of the rat calvarium. The cells were assayed after exposure to a field for 1-, 3-, 5-, 7-, and 10-day periods. Cells were exposed to north and south poles with a pole-face flux density of 0.61 T; control cultures were exposed to an unmagnetised piece of neodymium. After sham exposure or exposure to the magnetic field, 50 microCuries/ml of culture media of isotope were added to the culture medium. The cultures were returned to an incubator for 6 h. Then, following centrifugation, the supernatant was assayed for radioactivity in a scintillation counter after addition of 3 ml of scintillation fluid. A statistically significant magnetic stimulation of turnover rate and synthesis of fibroblasts was found, but stimulation of osteoblasts did not occur. Conversely, the explants, which represent the osteoblasts and fibroblasts in an organised system, showed a statistically significant inhibition in uptake of the radioactive label. The data indicate both variability and diversity of cellular behaviour, and they accentuate the need for caution in the interpretation of effects of static magnetic fields. PMID:8323569

  2. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    SciTech Connect

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.

  3. Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts

    SciTech Connect

    Dubaybo, B.A.; Thet, L.A. )

    1990-09-01

    The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either (3H)glucosamine or (35S)sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

  4. Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast

    PubMed Central

    Qu, Ling; Zhu, Rui; Li, Hongguo; Xue, Yingsen; Liu, Xincheng

    2016-01-01

    Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration. PMID:27525012

  5. Mesenchymal Stromal Cells but Not Cardiac Fibroblasts Exert Beneficial Systemic Immunomodulatory Effects in Experimental Myocarditis

    PubMed Central

    Miteva, Kapka; Pappritz, Kathleen; Westermann, Dirk; Schefold, Joerg C.; Fusch, Gerhard; Weithäuser, Alice; Rauch, Ursula; Becher, Peter-Moritz; Klingel, Karin; Ringe, Jochen; Kurtz, Andreas; Schultheiss, Heinz-Peter; Tschöpe, Carsten

    2012-01-01

    Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs. PMID:22815907

  6. Effect of Nd:YAG Low Level Laser Therapy on Human Gingival Fibroblasts

    PubMed Central

    Gkogkos, Andreas S.; Karoussis, Ioannis K.; Prevezanos, Ioannis D.; Marcopoulou, Kleopatra E.; Kyriakidou, Kyriaki; Vrotsos, Ioannis A.

    2015-01-01

    Aim. To evaluate the effect of Low Level Laser Therapy (LLLT) on human gingival fibroblasts in terms of proliferation and growth factors' secretion (EGF, bFGF, and VEGF). Materials and Methods. Primary cultures of keratinized mucosa fibroblasts were irradiated by a Nd:YAG laser 1064 nm with the following energy densities: 2.6 J/cm2, 5.3 J/cm2, 7.9 J/cm2, and 15.8 J/cm2. Controls were not irradiated. Cultures were examined for cell proliferation and growth factors' secretion after 24, 48, and 72 hours. All experimental procedures were performed in duplicate. Data were analyzed by Student's t-test (p < 0.05). Results. All laser-irradiation doses applied promoted a higher cell proliferation at 48 hours in a dose-response relationship compared to controls. This difference reached statistical significance for the cultures receiving 15.8 J/cm2 (p = 0.03). Regarding EGF, all laser irradiation doses applied promoted a higher secretion at 48 hours in a reverse dose-response pattern compared to controls. This difference reached statistical significance for the cultures receiving 2.6 J/cm2 (p = 0.04). EGF levels at the other time points, bFGF, and VEGF showed a random variation between the groups. Conclusion. Within the limits of this study, LLLT (Nd:YAG) may induce gingival fibroblasts' proliferation and upregulate the secretion of EGF. Further studies are needed to confirm these results. PMID:26504463

  7. Protective effect of selenium and zinc on UV-A damage in human skin fibroblasts.

    PubMed

    Leccia, M T; Richard, M J; Beani, J C; Faure, H; Monjo, A M; Cadet, J; Amblard, P; Favier, A

    1993-10-01

    Ultraviolet A radiation participates in cytotoxicity and carcinogenesis of the skin by a mechanism involving the generation of reactive oxygen species. Endogenous antiradical defense systems utilize metalloenzymes including Se-dependent glutathione peroxidase and Cu and Zn superoxide dismutase. The aim of the present work was to determine the protective effect of two trace elements, Se and Zn, on cultured human diploid fibroblasts exposed to UV-A radiation (broad-spectrum source with a maximum intensity at 375 nm). Selenium in the culture medium (0.1 mg/L) in the form of sodium selenite increased the synthesis and activity of glutathione peroxidase by 60.5% in the absence of exposure to UV-A radiation and by 35% after irradiation with 5 J/cm2 (P = 0.043). The presence of this element significantly increased the survival of UV-A-irradiated fibroblasts (P < 0.0001). This confirms the essential role of Se in the detoxifying activity of the enzyme. In addition, thiobarbituric acid-reacting substances (TBAR), which are lipid peroxidation markers, decreased in the presence of exogenous Se: -19% and -22% without irradiation and after irradiation with 5 J/cm2 (P = 0.056). When Zn was added at the dose of 6.5 mg/L as ZnCl2, fibroblasts subjected to oxidizing stress induced by UV-A were protected from cytotoxicity (P < 0.0001). The TBAR production decreased significantly: -33% without irradiation and -34% after irradiation with 5 J/cm2 (P = 0.008). Superoxide dismutase activity, however, decreased after supplementing with Zn: -26% without irradiation and -20% after UV-A irradiation (P = 0.017). The antioxidant properties of Zn are thus apparently independent of superoxide dismutase activity. PMID:8248330

  8. Antioxidant effects of the sarsaparilla via scavenging of reactive oxygen species and induction of antioxidant enzymes in human dermal fibroblasts.

    PubMed

    Park, Gunhyuk; Kim, Tae-mi; Kim, Jeong Hee; Oh, Myung Sook

    2014-07-01

    Ultraviolet (UV) radiation from sunlight causes distinct changes in collagenous skin tissues as a result of the breakdown of collagen, a major component of the extracellular matrix. UV irradiation downregulates reactive oxygen species (ROS)-elimination pathways, thereby promoting the production of ROS, which are implicated in skin aging. Smilax glabra Roxb (sarsaparilla) has been used in folk medicine because of its many effects. However, no study on the protective effects of sarsaparilla root (SR) on human dermal fibroblasts has been reported previously. Here, we investigated the protective effect of SR against oxidative stress in dermal fibroblasts. SR significantly inhibited oxidative damage and skin-aging factor via mitogen-activated protein kinase signaling pathways. Also, SR decreased Ca(2+) and ROS, mitochondrial membrane potential, dysfunction, and increased glutathione, NAD(P)H dehydrogenase and heme oxygenase-1. These results demonstrate that SR can protect dermal fibroblasts against UVB-induced skin aging via antioxidant effects. PMID:25022355

  9. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast.

    PubMed

    Seol, Ja Young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin's elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity. PMID:27194933

  10. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    PubMed Central

    Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin’s elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity. PMID:27194933

  11. Effects of extremely low frequency electromagnetic fields on human fetal scleral fibroblasts.

    PubMed

    Zhu, Huang; Wang, Jie; Cui, Jiefeng; Fan, Xianqun

    2016-06-01

    This study investigated the effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human fetal scleral fibroblasts (HFSFs). HFSFs were subjected to 50 Hz artificial ELF-EMFs generated by Helmholtz coils with 0.1, 0.2, 0.5, and 1.0 mT field intensities for 6 to 48 h. The viability and factors involved in scleral structuring of HFSFs were determined. The growth rate of HFSFs significantly decreased after only 24 h of exposure to ELF-EMFs (0.2 mT). The messenger RNA (mRNA) expression of collagen type I (COL1A1) decreased and expression of matrix metalloproteinase-2 (MMP-2) increased significantly. There was a decrease in tissue inhibitor of MMP-2 mRNA levels between treated and control cells only at the 1.0 mT intensity level. Transforming growth factor beta-2 mRNA increased in exposed cells, and, simultaneously, fibroblast growth factor-2 mRNA levels decreased. The protein expressions of COL1A1 and MMP-2 were also significantly altered subsequent to exposure (p < 0.05). This study shows that ELF-EMFs had biological effects on HFSFs and could cause abnormality in scleral collagen. PMID:25147305

  12. Smac127 Has Proapoptotic and Anti-Inflammatory Effects on Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

    PubMed

    Lattuada, D; Gualtierotti, R; Crotta, K; Seneci, P; Ingegnoli, F; Corradini, C; Viganò, R; Marelli, O; Casnici, C

    2016-01-01

    Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia. Fibroblast-like synoviocytes (FLSs) are apoptosis-resistant and contribute to the pathogenesis of RA by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. We evaluated the proapoptotic and anti-inflammatory activity of the small molecule Smac127 on RA-FLSs cultured in synovial fluid (SF), in order to reproduce the physiopathological environmental characteristic of RA joints. In this context, Smac127 induces apoptosis by inhibiting apoptosis proteins (IAPs). This inhibition activates caspase 3 and restores the apoptotic pathway. In addition, Smac127 induces a significant inhibition of the secretion of IL-15 and IL-6, stimulation of pannus formation, and damage of bone and cartilage in RA. Also the secretion of the anti-inflammatory cytokine IL-10 is dramatically increased in the presence of Smac127. The cartilage destruction in RA patients is partly mediated by metalloproteinases; here we show that the MMP-1 production by fibroblasts cultured in SF is significantly antagonized by Smac127. Conversely, this molecule has no significant effects on RANKL and OPG production. Our observations demonstrate that Smac127 has beneficial regulatory effects on inflammatory state of RA-FLSs and suggest a potential use of Smac127 for the control of inflammation and disease progression in RA. PMID:26989333

  13. Investigation of the phototoxic effect of ZnO nanorods on fibroblasts and melanoma human cells

    NASA Astrophysics Data System (ADS)

    Kishwar, S.; Siddique, M.; Israr-Qadir, M.; Nur, O.; Willander, M.; Öllinger, K.

    2014-11-01

    Photocytotoxic effects of as-grown and zinc oxide (ZnO) nanorods coated with 5-aminolevulinic acid (ALA) have been studied on human cells, i.e. melanoma and foreskin fibroblast, under dark and ultraviolet light exposures. Zinc oxide nanorods have been grown on the very sharp tip (diameter = 700 nm) of borosilicate glass pipettes and then were coated by the photosensitizer for targeted investigations inside human cells. The coated glass pipette’s tip with photosensitizer has been inserted inside the cells with the help of a micro-manipulator and irradiated through ultraviolet light (UVA), which reduces the membrane potential of the mitochondria leading to cell death. Cell viability loss has been detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay when exposed to the dissolved ZnO nanorods and the production of the reactive oxygen species (ROS) has been detected along with the enhanced cytotoxic effect under UVA irradiation. Additionally, the influence of the lipid soluble antioxidant vitamin E and water-soluble N-acetyl-cysteine toward the enhancement or reduction of the toxicity has been investigated. A comparative analysis of the toxic nature of ZnO nanorods has been drawn between normal human fibroblast and melanoma cells, which can be favorable for understanding the clinical setting for killing tumor cells.

  14. Differential effect of medium potent nonhalogenated double-ester-type and conventional glucocorticoids on proliferation and chemotaxis of fibroblasts in vitro.

    PubMed

    Hein, R; Korting, H C; Mehring, T

    1994-01-01

    Recently several attempts have been made to develop glucocorticoids which show less pronounced side effects. We intended, therefore, to use experimental systems to examine the influence of newly developed nonhalogenated glucocorticoids and conventional fluorinated congeners, demonstrating similar anti-inflammatory activity in vivo, on biosynthetic capacities of human fibroblasts. Fibroblasts were kept in monolayer cultures and exposed to different concentrations of the active compounds for 6 days to analyze the influence on proliferation. Chemotaxis of fibroblasts was studied in blind well Boyden chambers. Fibroblast-conditioned medium was used as chemoattractant. All glucocorticoids tested influenced fibroblast proliferation at high (10(-5) M) and low (10(-9) M) concentration. Yet the effect was clearly more marked with fluorinated compounds. Basically, the same applied for chemotaxis. At the low concentration, however, nonfluorinated glucocorticoids exerted almost no influence. These results suggest that modifications of steroidal structure can specifically influence their effects on biosynthetic capacities of fibroblasts. PMID:8054213

  15. The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

    PubMed Central

    Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

    2014-01-01

    Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

  16. Cytotoxic effects of mineral trioxide aggregate, calcium enrichedmixture cement, Biodentine and octacalcium pohosphate onhuman gingival fibroblasts.

    PubMed

    A Saberi, Eshagh; Farhadmollashahi, Narges; Ghotbi, Faroogh; Karkeabadi, Hamed; Havaei, Roholla

    2016-01-01

    Background. This in vitro study compared the effects of mineral trioxide aggregate (MTA), calcium enriched mixture(CEM) cement, Biodentine (BD) and octacalcium phosphate (OCP) on the viability of human gingival fibroblasts (HGFs). Methods. After completion of the setting time of the materials under study, fibroblasts were placed in 24-well insert platesand 1 mg of each material was added to the respective wells. The plates were then incubated at 37°C. The inserts were removedat 24, 48 and 168 hours and 2,5-diphenyltetrazolium bromide was added to assess cytotoxicity via the MTT colorimetricassay. Data were analyzed at different time intervals using repeated-measures ANOVA, followed by the Bonferronitest at three levels of significance of P < 0.05, P < 0.01 and P < 0.001. Results. Cytotoxicity of the materials under study was not significantly different at 24 and 48 hours compared to the controlgroup. However, at 168 hours, a significant difference was noted between MTA (P < 0.05) and Biodentine (P < 0.01)and the control group. Conclusion. Cytotoxicity of MTA, CEM, Biodentine and OCP against HGFs was similar to that of the control group at 24and 48 hours. Over time, MTA and Biodentine exhibited less cytotoxicity than other materials. PMID:27429722

  17. Secreted gingipains from Porphyromonas gingivalis colonies exert potent immunomodulatory effects on human gingival fibroblasts.

    PubMed

    Bengtsson, Torbjörn; Khalaf, Atika; Khalaf, Hazem

    2015-09-01

    Periodontal pathogens, including Porphyromonas gingivalis, can form biofilms in dental pockets and cause inflammation, which is one of the underlying mechanisms involved in the development of periodontal disease, ultimately leading to tooth loss. Although P. gingivalis is protected in the biofilm, it can still cause damage and modulate inflammatory responses from the host, through secretion of microvesicles containing proteinases. The aim of this study was to evaluate the role of cysteine proteinases in P. gingivalis colony growth and development, and subsequent immunomodulatory effects on human gingival fibroblast. By comparing the wild type W50 with its gingipain deficient strains we show that cysteine proteinases are required by P. gingivalis to form morphologically normal colonies. The lysine-specific proteinase (Kgp), but not arginine-specific proteinases (Rgps), was associated with immunomodulation. P. gingivalis with Kgp affected the viability of gingival fibroblasts and modulated host inflammatory responses, including induction of TGF-β1 and suppression of CXCL8 and IL-6 accumulation. These results suggest that secreted products from P. gingivalis, including proteinases, are able to cause damage and significantly modulate the levels of inflammatory mediators, independent of a physical host-bacterial interaction. This study provides new insight of the pathogenesis of P. gingivalis and suggests gingipains as targets for diagnosis and treatment of periodontitis. PMID:26302843

  18. Cytotoxic effects of mineral trioxide aggregate, calcium enrichedmixture cement, Biodentine and octacalcium pohosphate onhuman gingival fibroblasts

    PubMed Central

    A. Saberi, Eshagh; Farhadmollashahi, Narges; Ghotbi, Faroogh; Karkeabadi, Hamed; Havaei, Roholla

    2016-01-01

    Background. This in vitro study compared the effects of mineral trioxide aggregate (MTA), calcium enriched mixture(CEM) cement, Biodentine (BD) and octacalcium phosphate (OCP) on the viability of human gingival fibroblasts (HGFs). Methods. After completion of the setting time of the materials under study, fibroblasts were placed in 24-well insert platesand 1 mg of each material was added to the respective wells. The plates were then incubated at 37°C. The inserts were removedat 24, 48 and 168 hours and 2,5-diphenyltetrazolium bromide was added to assess cytotoxicity via the MTT colorimetricassay. Data were analyzed at different time intervals using repeated-measures ANOVA, followed by the Bonferronitest at three levels of significance of P < 0.05, P < 0.01 and P < 0.001. Results. Cytotoxicity of the materials under study was not significantly different at 24 and 48 hours compared to the controlgroup. However, at 168 hours, a significant difference was noted between MTA (P < 0.05) and Biodentine (P < 0.01)and the control group. Conclusion. Cytotoxicity of MTA, CEM, Biodentine and OCP against HGFs was similar to that of the control group at 24and 48 hours. Over time, MTA and Biodentine exhibited less cytotoxicity than other materials. PMID:27429722

  19. Effect of Vascular Endothelial Growth Factor and Erythropoietin on Functional Activity of Fibroblasts and Multipotent Mesenchymal Stromal Cells.

    PubMed

    Bondarenko, N A; Nikonorova, Yu V; Surovtseva, M A; Lykov, A P; Poveshchenko, O V; Poveshchenko, A F; Pokushalov, E A; Romanov, A B; Konenkov, V I

    2016-02-01

    The study examined the effect of VEGF and erythropoietin on proliferative and migratory activities of skin fibroblasts and multipotent mesenchymal stromal cells of human adipose tissue. VEGF stimulated proliferation and migration of fi broblasts, but produced no significant effect on functional activity of multipotent mesenchymal stem cells. Erythropoietin stimulated proliferation of both cell types, but did not affect their migration. PMID:26899850

  20. Effects of Simulated Microgravity on Sensitivity of Human Fibroblasts to Radiation

    NASA Technical Reports Server (NTRS)

    Whitehead, Nickolas

    2016-01-01

    Living organisms are exposed to radiation in space that consists of high energy protons and heavy charged particles. For humans, exposure to this environment is expected to cause cancer and other harmful effects. Current assessment of space radiation risk to astronauts is based on the information gained from human data and animal experiments under 1g gravity. If spaceflight factors, such as microgravity, affect the repair of space radiation-induced damage, then one would expect an additional impact on the mutation rate in living cells and consequently on the accuracy of current ground-based risk assessment methods. The project I worked on consisted of using clonogenic assays to analyze the survival of human fibroblast AG01522 cells exposed to radiation with and without simulated microgravity. A random positioning machine (RPM) was used to simulate microgravity because of the principle of gravity-vector-averaging. The effects of simulated microgravity were studied after exposing the cells to different doses of gamma radiation.

  1. Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

    PubMed Central

    Choi, Min Ji; Song, Kang-Moon; Park, Jin-Mi; Kwon, Mi-Hye; Kwon, Ki-Dong; Park, Soo-Hwan; Ryu, Dong-Soo; Ryu, Ji-Kan; Suh, Jun-Kyu

    2015-01-01

    Transforming growth factor-β1 (TGF-β1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD. PMID:25532569

  2. Bioremediation of hexavalent chromate using permeabilized Brevibacterium sp. and Stenotrophomonas sp. cells.

    PubMed

    Ge, Shimei; Ge, Shichao; Zhou, Maohong; Dong, Xinjiao

    2015-07-01

    Bioremediation has been found to be a useful method for removing hexavalent chromium (Cr(VI)), which is very toxic, from wastewater. Two strains of bacteria that were able to reduce Cr(VI) effectively were isolated from Cr(VI) contaminated soil samples and identified as Brevibacterium sp. K1 and Stenotrophomonas sp. D6, respectively, based on 16S rRNA gene sequence analyses. Brevibacterium sp. K1 and Stenotrophomonas sp. D6 could grow in Luria-Broth medium containing K2Cr2O7 at 1000 and 1600 mg/L, respectively, and they completely reduced the Cr(VI) in LB medium containing K2Cr2O7 at 200 mg/L within 72 h. Further analyses revealed that permeabilized K1 and D6 cells reduced Cr(VI) more effectively than did the resting cells. Triton X-100 was the best permeabilizing agent that was tested. The permeabilized cells of both strains could completely reduce Cr(VI) in industrial wastewater twice before needing to be replenished. The results suggested that these chromate-reducing bacteria are potential candidates for practical use biotreating industrial effluents containing Cr(VI) with Stenotrophomonas sp. D6 being the more effective bacterium. PMID:25881152

  3. Effects of alendronate and pamidronate on apoptosis and cell proliferation in cultured primary human gingival fibroblasts.

    PubMed

    Soydan, S S; Araz, K; Senel, F V; Yurtcu, E; Helvacioglu, F; Dagdeviren, A; Tekindal, M A; Sahin, F

    2015-11-01

    Data arising from the recent literature directed the researchers to study on the degree and extent of bisphosphonate toxicity on oral mucosa in further detail. The aim of this study is to determine the half maximal inhibitory concentration of pamidronate (PAM) and alendronate (ALN) on human gingival fibroblasts in vitro using 3-[4.5-thiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) assay and to evaluate the effects of both agents on the proliferation and apoptotic indices. Cells used in the study were generated from human gingival specimens and divided into alendronate (n = 240), PAM (n = 240), and control groups (n = 60). Based on the MTT assay results, 10(-4), 10(-5), 10(-6), and 10(-7) M concentrations of both drugs were administered and the effects were evaluated for 6, 12, 24, 48, or 72 h periods. An indirect immunofluorescence technique was used to evaluate apoptotic (anti-caspase 3) and proliferation (anti-Ki67) indices. Toxicity of both PAM and ALN was found to be the most potent at 10(-4)-10(-5) M range. The apoptotic index of PAM group was found to be significantly higher than ALN group for all concentrations especially at 24 h incubation time (p < 0.05). The decrease in the proliferation index was found similar in first 48 h for both drugs; however, after 72 h of incubation decrease in proliferation index in PAM group was found to be significantly higher (p < 0.05). Micromolar concentrations of not only PAM but also ALN rapidly affect cells generated from human oral gingival tissue by inducing apoptosis together with inhibition of proliferation. Cytotoxic effects of both ALN and PAM on primary human gingival fibroblasts, which cause significant changes in apoptotic and proliferative indices as shown in this in vitro study, suggests that the defective epithelialization of oral mucosa is possibly a major factor on the onset of bisphosphonate-related osteonecrosis of the jaw cases. PMID:25636638

  4. Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

    PubMed Central

    Levi-Schaffer, F; Segal, V; Shalit, M

    1991-01-01

    We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release. PMID:2016117

  5. Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells.

    PubMed

    Garcia, Lucas da Fonseca Roberti; Santos, Alailson Domingos dos; Moraes, João Carlos Silos; Costa, Carlos Alberto de Souza

    2016-01-01

    The present study aimed at evaluating the cytotoxic effects of a novel cement called CER on periodontal fibroblast-like cells of mice (MDPL-20), in comparison with different formulations of Mineral Trioxide Aggregate (MTA), by means of the cell viability test (MTT) and cell morphology analysis. Thirty-two round-shaped samples were fabricated with the following cements: white MTA, white and gray CER and experimental white MTA. The samples were immersed in serum-free culture medium for 24 hours or 7 days (n = 16). The extracts (culture medium + components released from the cements) were applied for 24 hours to previously cultured cells (40.000 cells/cm2) in the wells of 24-well plates. Cells seeded in complete culture medium were used as a negative control. Cell viability was assessed using the MTT assay. Two samples of each cement were used for cell morphology analysis by Scanning Electron Microscopy (SEM). The extracts obtained at the 7-day period presented higher cytotoxicity compared with the 24-hour period (p < 0.05). The gray CER obtained at 24 hours presented the highest cytotoxic effect, whereas the experimental white MTA presented the lowest, similar to the control (p > 0.05). However, at the 7-day period, the experimental white MTA presented no significant difference in comparison with the other cements (p > 0.05). At the 7-day period, CER cement presented cytotoxic effects on fibroblast-like cells, similar to different MTA formulations. However, the immersion period in the culture medium influenced the cytotoxicity of the cements, which was greater for CER cement at 24 hours. PMID:26981755

  6. The effect of pantothenic acid deficiency on keratinocyte proliferation and the synthesis of keratinocyte growth factor and collagen in fibroblasts.

    PubMed

    Kobayashi, Daisaku; Kusama, Miho; Onda, Masaaki; Nakahata, Norimichi

    2011-01-01

    It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol derivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. PMID:21258175

  7. A Novel Protocol for Decoating and Permeabilizing Bacterial Spores for Epifluorescent Microscopy

    NASA Technical Reports Server (NTRS)

    LaDuc, Myron T.; Mohapatra, Bidyut

    2014-01-01

    Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 µL of 100 mM Tris-HCl, 30 µL of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 ºC. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 ºC for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (˜50

  8. Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

    PubMed

    Ershova, E S; Sergeeva, V A; Chausheva, A I; Zheglo, D G; Nikitina, V A; Smirnova, T D; Kameneva, L V; Porokhovnik, L N; Kutsev, S I; Troshin, P A; Voronov, I I; Khakina, E A; Veiko, N N; Kostyuk, S V

    2016-07-01

    Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo. PMID:27402482

  9. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

    SciTech Connect

    Pardo-Andreu, Gilberto L.; Tudella, Valeria G.

    2011-06-15

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 {mu}M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca{sup 2+} efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP{sup +} transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: > We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. > GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. > These actions

  10. Effects of different forms of chitosan on intercellular junctions of mouse fibroblasts in vitro.

    PubMed

    Uslu, B; Biltekin, B; Denir, S; Özbaş-Turan, S; Arbak, S; Akbuğa, J; Bilir, A

    2016-01-01

    Chitosan is a linear polysaccharide that has many biomedical applications. We compared the effects of chitosan, in both solution and membranous form, on intercellular adhesion of Swiss 3T3 mouse fibroblasts. Cells were grown as spheroidal cell cultures. Some control cell spheroids were cultured without chitosan and two experimental groups were cultured with chitosan. Chitosan in solution was used for one experimental group and chitosan in membranous form was used for the other. For each group, intercellular adhesion was investigated on days 5 and 10 of culture. Transmission electron microscopy revealed well-defined cellular projections that were more prominent in cells exposed to either membranous or solution forms of chitosan than to the chitosan-free control. Immunocytochemical staining of ICAM-1 and e-cadherin was used to determine the development of intercellular junctions. Compared to the weakly stained control, strong reactions were observed in both chitosan exposed groups at both 5 and 10 days. Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and incubated with anti-BrdU primary antibody to assess proliferation. Both the solution and membranous forms of chitosan increased proliferation at both 5 and 10 days. Cellular viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The MTT assay indicated high cell viability; maximum viability was obtained with the solution form of chitosan at day 5. Chitosan exposure increased the number of intercellular junctions and showed a significant proliferative effect on 3T3 mouse fibroblasts. PMID:26523482

  11. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

    PubMed Central

    Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-01

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

  12. Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

    SciTech Connect

    Voelker, D.R. )

    1989-12-01

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({sup 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.

  13. Increased effectiveness of carbon ions in the production of reactive oxygen species in normal human fibroblasts

    PubMed Central

    Dettmering, Till; Zahnreich, Sebastian; Colindres-Rojas, Miriam; Durante, Marco; Taucher-Scholz, Gisela; Fournier, Claudia

    2015-01-01

    The production of reactive oxygen species (ROS), especially superoxide anions (O2·–), is enhanced in many normal and tumor cell types in response to ionizing radiation. The influence of ionizing radiation on the regulation of ROS production is considered as an important factor in the long-term effects of irradiation (such as genomic instability) that might contribute to the development of secondary cancers. In view of the increasing application of carbon ions in radiation therapy, we aimed to study the potential impact of ionizing density on the intracellular production of ROS, comparing photons (X-rays) with carbon ions. For this purpose, we used normal human cells as a model for irradiated tissue surrounding a tumor. By quantifying the oxidization of Dihydroethidium (DHE), a fluorescent probe sensitive to superoxide anions, we assessed the intracellular ROS status after radiation exposure in normal human fibroblasts, which do not show radiation-induced chromosomal instability. After 3–5 days post exposure to X-rays and carbon ions, the level of ROS increased to a maximum that was dose dependent. The maximum ROS level reached after irradiation was specific for the fibroblast type. However, carbon ions induced this maximum level at a lower dose compared with X-rays. Within ∼1 week, ROS decreased to control levels. The time-course of decreasing ROS coincides with an increase in cell number and decreasing p21 protein levels, indicating a release from radiation-induced growth arrest. Interestingly, radiation did not act as a trigger for chronically enhanced levels of ROS months after radiation exposure. PMID:25304329

  14. Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

    PubMed Central

    Patel, Hiren; Huynh, Quang; Bärlehner, Dominik; Heerklotz, Heiko

    2014-01-01

    Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C12EO8. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C12EO8. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection. PMID:24853740

  15. Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure.

    PubMed

    Thompson, Gary L; Roth, Caleb C; Kuipers, Marjorie A; Tolstykh, Gleb P; Beier, Hope T; Ibey, Bennett L

    2016-01-29

    Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus. PMID:26721436

  16. Influence of laser parameters on nanoparticle-induced membrane permeabilization

    NASA Astrophysics Data System (ADS)

    Yao, Cuiping; Qu, Xiaochao; Zhang, Zhenxi; Hüttmann, Gereon; Rahmanzadeh, Ramtin

    2009-09-01

    Light-absorbing nanoparticles that are heated by short laser pulses can transiently increase membrane permeability. We evaluate the membrane permeability by flow cytometry assaying of propidium iodide and fluorescein isothiocyanate dextran (FITC-D) using different laser sources. The dependence of the transfection efficiency on laser parameters such as pulse duration, irradiant exposure, and irradiation mode is investigated. For nano- and also picosecond irradiation, we show a parameter range where a reliable membrane permeabilization is achieved for 10-kDa FITC-D. Fluorescent labeled antibodies are able to penetrate living cells that are permeabilized using these parameters. More than 50% of the cells are stained positive for a 150-kDa IgG antibody. These results suggest that the laser-induced permeabilization approach constitutes a promising tool for targeted delivery of larger exogenous molecules into living cells.

  17. The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    Progressive lowering of pH is characteristic for the endocytic pathway and enables efficient degradation of molecules by hydrolytic enzymes at its distal end. The existence of the proton gradient over the endosomal/lysosomal membranes depends on the action of the vacuolar ATPase (v-ATPase). During lysosomal membrane permeabilization (LMP), protons leak through the destabilized membrane, resulting in loss of the pH gradient. Here, we present a protocol showing how this effect can be detected by staining cells with lysosomotropic dyes, which accumulate in acidic organelles after protonation. During LMP, cells lose the ability to retain these dyes and therefore appear pale. Among the most commonly used lysosomotropic dyes are LysoTracker reagents and acridine orange. Cells can be analyzed with a fluorescence microscope; however, flow-cytometric analysis enables fast, objective, and reliable evaluation of differences between samples. Advantages of the technique include the fact that sample preparation is relatively simple and can be scaled-up to test several different compounds or conditions. However, as we will discuss, cells treated with v-ATPase inhibitors also lose the pH gradient across lysosomal membranes and cannot be stained with lysosomotropic dyes, although this is not accompanied by LMP. Therefore, merely observing loss of staining is not in itself a proof of LMP. PMID:27140914

  18. Effect of tripeptide-copper complexes on the process of skin wound healing and on cultured fibroblasts.

    PubMed

    Buffoni, F; Pino, R; Dal Pozzo, A

    1995-01-01

    The effects of Gly-His-Lys-Cu and of three synthetic analogues (I, II and III) on wound healing of the guinea-pig dorsal skin, as well as on cultured fibroblasts, were examined. Gly-His-Lys-Cu and peptide I-Cu were tested in vivo. Hydroxyproline, proteins, DNA and semicarbazide-sensitive amine oxidase, with a high affinity for benzylamine, were measured, and the histology of the wounds was observed after staining with hematoxylin/eosin. Another set of wounds was treated in parallel with equivalent amounts of copper acetate. Gly-His-Lys-Cu and the analogues caused a decrease of the activity of semicarbazide-sensitive amine oxidase, with a high affinity for benzylamine, 4-8 days after surgery, followed by an increase on day 11 that was higher than in the control group. No significant difference was found between the two peptides. A slower reorganization of the skin and a delayed activation of fibroblasts are the main effects observed with these peptides-Cu complexes. Preliminary studies on cultured fibroblasts were monitored to see whether these peptides had a direct effect on fibroblasts. The products studied at a concentration of 10(-7) M, decreased cell reproduction and increased collagen expression. PMID:8836453

  19. Protective Effect of Processed Panax ginseng, Sun Ginseng on UVB-irradiated Human Skin Keratinocyte and Human Dermal Fibroblast

    PubMed Central

    Lee, Hyejin; Lee, Joo Yeop; Song, Kyu Choon; Kim, Jinhee; Park, Jeong Hill; Chun, Kwang-Hoon; Hwang, Gwi Seo

    2012-01-01

    In this study, we investigated the protective effects of processed Panax ginseng, sun ginseng (SG) against the UVB-irradiation on epidermal keratinocytes and dermal fibroblasts. Pretreatment of SG in HaCaT keratinocytes and human dermal fibroblasts reduced UVB-induced cell damage as seen by reduced lactate dehydrogenase release. We also found that SG restored the UVB-induced decrease in anti-apoptotic gene expression (bcl-2 and bcl-xL) in these cells, indicating that SG has an anti-apoptotic effect and thus can protect cells from cell death caused by strong UVB radiation. In addition, SG inhibited the excessive expression of c-jun and c-fos gene by the UVB in HeCaT cells and human dermal fibroblasts. We also demonstrated that SG may exert an anti-inflammatory activity by reducing the nitric oxide production and inducible nitric oxide synthase mRNA synthesis in HaCaT keratinocytes and human dermal fibroblasts. This was further supported by its inhibitory effects on the elevated cyclooxygenase-2 and tumor necrosis factor-α transcription which was induced by UVB-irradiation in HaCaT cells. In addition, SG may have anti-aging property in terms of induction of procollagen gene expression and inhibition of the matrix metalloprotease-1 gene expression caused by UVBexposure. These findings suggest that SG can be a potential agent that may protect against the dermal cell damage caused by UVB. PMID:23717106

  20. Assessment of the Contractile Properties of Permeabilized Skeletal Muscle Fibers.

    PubMed

    Claflin, Dennis R; Roche, Stuart M; Gumucio, Jonathan P; Mendias, Christopher L; Brooks, Susan V

    2016-01-01

    Permeabilized individual skeletal muscle fibers offer the opportunity to evaluate contractile behavior in a system that is greatly simplified, yet physiologically relevant. Here we describe the steps required to prepare, permeabilize and preserve small samples of skeletal muscle. We then detail the procedures used to isolate individual fiber segments and attach them to an experimental apparatus for the purpose of controlling activation and measuring force generation. We also describe our technique for estimating the cross-sectional area of fiber segments. The area measurement is necessary for normalizing the absolute force to obtain specific force, a measure of the intrinsic force-generating capability of the contractile system. PMID:27492182

  1. Histological Effect of Basic Fibroblast Growth Factor on Chronic Vocal Fold Scarring in a Rat Model

    PubMed Central

    Tateya, Ichiro; Tateya, Tomoko; Sohn, Jin-Ho; Bless, Diane M.

    2016-01-01

    Objectives Vocal fold scarring is one of the most challenging laryngeal disorders to treat and there are currently no consistently effective treatments available. Our previous studies have shown the therapeutic potential of basic fibroblast growth factor (bFGF) for vocal fold scarring. However, the histological effects of bFGF on scarred vocal fold have not been elucidated. The aim of this study was to examine the histological effects of bFGF on chronic vocal fold scarring. Methods Sprague-Dawley rats were divided into phosphate buffered saline (sham) and bFGF groups. Unilateral vocal fold stripping was performed and the drug was injected into the scarred vocal fold for each group 2 months postoperatively. Injections were performed weekly for 4 weeks. Two months after the last injection, larynges were harvested and histologically analyzed. Results A significant increase of hyaluronic acid was observed in the vocal fold of the bFGF group compared with that of the sham group. However, there was no remarkable change in collagen expression nor in vocal fold contraction. Conclusion Significant increase of hyaluronic acid by local bFGF injection was thought to contribute to the therapeutic effects on chronic vocal fold scarring. PMID:26976028

  2. Effects of sphingomyelin and phosphatidylcholine degradation on cyclodextrin-mediated cholesterol efflux in cultured fibroblasts.

    PubMed

    Ohvo, H; Olsio, C; Slotte, J P

    1997-11-15

    The hydrolysis of plasma membrane sphingomyelin is known to dramatically alter cellular cholesterol homeostasis in different ways, whereas the degradation of plasma membrane phosphatidylcholine has much less or no effects on cell cholesterol homeostasis [Pörn, Ares, Slotte, J. Lipid Res. 34 (1993) 1385-1392]. In this study, we used an efficient extracellular cholesterol acceptor (cyclodextrin) and determined the extent of cholesterol efflux from cultured fibroblasts in which plasma membrane sphingomyelin or phosphatidylcholine was degraded. Treatment of cells with sphingomyelinase reduced the cell sphingomyelin content by about 76% (about 13 nmol SM degraded), and dramatically increased the desorption of [3H]cholesterol from the plasma membrane to 2-hydroxypropyl-beta-cyclodextrin. The corresponding hydrolysis of cell surface phosphatidylcholine (about 12% reduction of the cellular phosphatidylcholine content, corresponding to about 12 nmol degraded PC) had almost no effect on cell [3H]cholesterol efflux. The stimulatory effect of sphingomyelin degradation on cell [3H]cholesterol efflux was reversible, since rates of [3H]cholesterol efflux dropped back to control levels when cells (in this case baby hamster kidney cells) were allowed to restore their sphingomyelin content by re-synthesis in the absence of sphingomyelinase. The findings of this study clearly demonstrate that plasma membrane sphingomyelin markedly affected the rate of cholesterol transfer between cells and an extracellular acceptor (i.e., cyclodextrin), whereas the effect of phosphatidylcholine on cholesterol efflux was much smaller. PMID:9421186

  3. Effect of botulinum neurotoxin type A (BoNTA) on the morphology and viability of 3T3 murine fibroblasts

    PubMed Central

    Bandala, Cindy; Terán-Melo, Juan Luis; Anaya-Ruiz, Maricruz; Mejía-Barradas, Cesar Miguel; Domínguez-Rubio, Rene; la Garza-Montano, Paloma De; Alfaro-Rodríguez, Alfonso; Lara-Padilla, Eleazar

    2015-01-01

    Aim: BoNTA is used in the treatment of ophthalmological disorders, muscular hyperactivity and pain. In recent years it has been described that BoNTA reduces cellular viability and induces apoptosis in prostate cells lines. Studies about the effect of BoNTA are no well known. There have been studies about the effect of BoNTA on the expression levels of collagenase in fibroblasts, but not on its morphological impact on these cells. The aim of this study was to determine the effect of BoNTA on the morphology and viability of the 3T3 fibroblast cell line. Material and methods: The 3T3 fibroblast cell line was cultured and the experimental group received 10 U BoNTA added to a 0.9% sterile saline solution in a reconstituted vial. The control group received saline solution only. Cultured cells were observed and photographed at 5, 10, 15 and 20 h. Cell viability was evaluated by means of the trypan blue test, and cell proliferation with the Proliferation Assay kit (PROMEGA). Results: The application of BoNTA to 3T3 fibroblast cells induced morphological changes, such as a loss of normal fibroblast morphology. Additionally, we observed the cytoplasmic retraction and spread phenomena. The nuclei showed other important changes with Giemsa staining. Conclusion: The results indicate that BoNTA induced a loss of spindle form, increase in cytoplasmic vesicles, and the presence of nuclear vesicles (compacted chromatin surrounded by a nuclear envelope). This suggests an apoptotic process and decreased cell viability. Further studies are needed to explore the mechanisms of these alterations. PMID:26464704

  4. Cytoprotective Effects of Grape Seed Extract on Human Gingival Fibroblasts in Relation to Its Antioxidant Potential

    PubMed Central

    Katsuda, Yusuke; Niwano, Yoshimi; Nakashima, Takuji; Mokudai, Takayuki; Nakamura, Keisuke; Oizumi, Satomi; Kanno, Taro; Kanetaka, Hiroyasu; Egusa, Hiroshi

    2015-01-01

    Cytoprotective effects of short-term treatment with grape seed extract (GSE) upon human gingival fibroblasts (hGFs) were evaluated in relation to its antioxidant properties and compared with those of a water-soluble analog of vitamin E: trolox (Tx). GSE and Tx showed comparable antioxidant potential in vitro against di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH; a stable radical), hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Pretreatment or concomitant treatment with GSE for 1 min protected hGFs from oxidative stressors, including H2O2, acid-electrolyzed water (AEW), and 1O2, and attenuated the intracellular formation of reactive oxygen species induced by H2O2 and AEW. Tx also reduced the H2O2- and AEW-induced intracellular formation of reactive oxygen species, but showed no cytoprotective effects on hGFs exposed to H2O2, AEW, or 1O2. These results suggest that the cytoprotective effects of GSE are likely exerted independently of its antioxidant potential. PMID:26258747

  5. Biological effects of in vitro THz radiation exposure in human foetal fibroblasts.

    PubMed

    De Amicis, Andrea; Sanctis, Stefania De; Cristofaro, Sara Di; Franchini, Valeria; Lista, Florigio; Regalbuto, Elisa; Giovenale, Emilio; Gallerano, Gian Piero; Nenzi, Paolo; Bei, Roberto; Fantini, Massimo; Benvenuto, Monica; Masuelli, Laura; Coluzzi, Elisa; Cicia, Cristina; Sgura, Antonella

    2015-11-01

    In recent years, terahertz (THz) radiation has been widely used in a variety of applications: medical, security, telecommunications and military areas. However, few data are available on the biological effects of this type of electromagnetic radiation and the reported results, using different genetic or cellular assays, are quite discordant. This multidisciplinary study focuses on potential genotoxic and cytotoxic effects, evaluated by several end-points, associated with THz radiation. For this purpose, in vitro exposure of human foetal fibroblasts to low frequency THz radiation (0.1-0.15THz) was performed using a Compact Free Electron Laser. We did not observe an induction of DNA damage evaluated by Comet assay, phosphorylation of H2AX histone or telomere length modulation. In addiction, no induction of apoptosis or changes in pro-survival signalling proteins were detected. Moreover, our results indicated an increase in the total number of micronuclei and centromere positive micronuclei induction evaluated by CREST analysis, indicating that THz radiation could induce aneugenic rather than clastogenic effects, probably leading to chromosome loss. Furthermore, an increase of actin polymerization observed by ultrastructural analysis after THz irradiation, supports the hypothesis that an abnormal assembly of spindle proteins could lead to the observed chromosomal malsegregation. PMID:26520385

  6. Photoprotective Effects of Cycloheterophyllin against UVA-Induced Damage and Oxidative Stress in Human Dermal Fibroblasts.

    PubMed

    Huang, Cheng-Hua; Li, Hsin-Ju; Wu, Nan-Lin; Hsiao, Chien-Yu; Lin, Chun-Nan; Chang, Hsun-Hsien; Hung, Chi-Feng

    2016-01-01

    Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H2O2) generation and down-regulate H2O2-induced MAPK phosphorylation. In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging. PMID:27583973

  7. The action of mimetic peptides on connexins protects fibroblasts from the negative effects of ischemia reperfusion

    PubMed Central

    Glass, Beverley J.; Hu, Rebecca G.; Phillips, Anthony R. J.; Becker, David L.

    2015-01-01

    ABSTRACT Connexins have been proposed as a target for therapeutic treatment of a variety of conditions. The main approaches have been by antisense or small peptides specific against connexins. Some of these peptides enhance communication while others interfere with connexin binding partners or bind to the intracellular and extracellular loops of connexins. Here, we explored the mechanism of action of a connexin mimetic peptide by evaluating its effect on gap junction channels, connexin protein levels and hemichannel activity in fibroblast cells under normal conditions and following ischemia reperfusion injury which elevates Cx43 levels, increases hemichannel activity and causes cell death. Our results showed that the effects of the mimetic peptide were concentration-dependent. High concentrations (100-300 μM) significantly reduced Cx43 protein levels and GJIC within 2 h, while these effects did not appear until 6 h when using lower concentrations (10-30 μM). Cell death can be reduced when hemichannel opening and GJIC were minimised. PMID:26471768

  8. Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture.

    PubMed

    Emonet-Piccardi, N; Richard, M J; Ravanat, J L; Signorini, N; Cadet, J; Béani, J C

    1998-10-01

    Ultraviolet A radiation (UVA, 320-400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 microM) or zinc chloride (100 microM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm2. In the Comet assay, the tail moment increased by 45% (1 J/cm2) to 89% (6J/cm2) in non-supplemented cells (p)<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm2 (p < 0.05). For the highest UVA dose (6J/cm2), Se and Zn were more effective than NAC (p < 0.01). PMID:9860045

  9. Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

    PubMed Central

    Keslová-Veselíková, Jana; Hůlková, Helena; Dobrovolný, Robert; Asfaw, Befekadu; Poupětová, Helena; Berná, Linda; Sikora, Jakub; Goláň, Lubor

    2008-01-01

    The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage. PMID:18351385

  10. Ultraviolet-B Protective Effect of Flavonoids from Eugenia caryophylata on Human Dermal Fibroblast Cells

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2015-01-01

    Background: The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. Objective: To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast cells. Materials and Methods: Protective ability of flavonoid-enriched (FE) fraction of clove was studied against UV-B induced cytotoxicity, anti-oxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2 antioxidant response element (Nrf2 ARE) pathway. Results: FE fraction showed a significant antioxidant potential. Pretreatment of cells with FE fraction (10–40 μg/ml) reversed the effects of UV-B induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: The present study demonstrated for the first time that the FE fraction from clove could confer UV-B protection probably through the Nrf2-ARE pathway, which included the down-regulation of Nrf2 and HO-1. These findings suggested that the flavonoids from clove could potentially be considered as UV-B protectants and can be explored further for its topical application to the area of the skin requiring protection. SUMMARY Pretreatment of human dermal fibroblast with flavonoid-enriched fraction of Eugenia caryophylata attenuated effects of ultraviolet-B radiationsIt also conferred protection through nuclear factor E2-related factor 2-antioxidant response pathway and increased tolerance of cells against oxidative stress

  11. Radiation induced bystander effect by GAP junction channels in human fibroblast cell

    NASA Astrophysics Data System (ADS)

    Furusawa, Y.; Shao, C.; Aoki, M.; Kobayashi, Y.; Funayama, T.; Ando, K.

    The chemical factor involved in bystander effect and its transfer pathway were investigated in a confluent human fibroblast cell (AG1522) population. Micronuclei (MN) and G1-phase arrest were detected in cells irradiated by carbon (~100 keV/μm) ions at HIMAC. A very low dose irradiation showed a high effectiveness in producing MN, suggesting a bystander effect. This effectiveness was enhanced by 8-Br-cAMP treatment that increases gap junctional intercellular communication (GJIC). On the other hand, the effect was reduced by 5% DMSO treatment, which reduce the reactive oxygen species (ROS), and suppressed by 100 μM lindane treatment, an inhibitor of GJIC. In addition, the radiation-induced G1-phase arrest was also enhanced by cAMP, and reduced or suppressed by DMSO or lindane. A microbeam device (JAERI) was also used for these studies. It was found that exposing one single cell in a confluent cell population to exactly one argon (~1260 keV/μm) or neon (~430 keV/ μm) ion, additional MN could be detected in many other unirradiated cells. The yield of MN increased with the number of irradiated cells. However, there was no significant difference in the MN induction when the cells were irradiated by increasing number of particles. MN induction by bystander effect was partly reduced by DMSO, and effectively suppressed by lindane. Our results obtained from both random irradiation and precise numbered irradiation indicate that both GJIC and ROS contributed to the radiation-induced bystander effect, but the cell gap junction channels likely play an essential role in the release and transfer of radiation-induced chemical factors.

  12. The effects of ascorbic acid and iron co-supplementation on the proliferation of 3T3 fibroblasts.

    PubMed

    Collis, C S; Yang, M; Peach, S J; Diplock, A T; Rice-Evans, C

    1996-07-01

    Exposure of 3T3 fibroblasts to FeII reveals a concentration-dependent inhibition of cell proliferation compared to control cells, the apparent threshold for this iron-mediated effect being 5 microM FeII. The inhibition of cell proliferation was accompanied by an enhancement of total malondialdehyde (MDA) levels (as detected directly by hplc) in the cells at higher iron concentrations. The co-supplementation of FeII with varying concentrations of ascorbic acid over the range 5 microM to 240 microM had no significant effect on the threshold for iron toxicity or lipid peroxidation. These results show that there is neither a significant exacerbation of the pro-oxidant effect of FeII nor any protective effect of ascorbate when cultures of 3T3 mouse fibroblasts are exposed to co-supplementation regimes of iron with ascorbic acid. PMID:8814446

  13. FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

    PubMed Central

    2013-01-01

    Background Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. Results We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. Conclusions Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential. PMID:24066673

  14. Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

    PubMed Central

    Domnina, L V; Gelfand, V I; Ivanova, O Y; Leonova, E V; Pletjushkina, O Y; Vasiliev, J M; Gelfand, I M

    1982-01-01

    The effects of low doses of cytochalasin B (2 micrograms/ml) and cytochalasin D (0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors. Images PMID:6961447

  15. Antioxidant effects of antioxidant biofactor on reactive oxygen species in human gingival fibroblasts

    PubMed Central

    Matsui, Satoshi; Tsujimoto, Yasuhisa; Ozawa, Toshihiko; Matsushima, Kiyoshi

    2011-01-01

    The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O2•−) and hydroxyl radical (•OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O2•− and •OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H2O2 (AOB + H2O2 group), containing H2O2 (H2O2 group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS. PMID:21562640

  16. Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells.

    PubMed

    Pan, Zhi; Lee, Wilson; Slutsky, Lenny; Clark, Richard A F; Pernodet, Nadine; Rafailovich, Miriam H

    2009-04-01

    The effects of exposure of human dermal fibroblasts to rutile and anatase TiO(2) nanoparticles are reported. These particles can impair cell function, with the latter being more potent at producing damage. The exposure to nanoparticles decreases cell area, cell proliferation, mobility, and ability to contract collagen. Individual particles are shown to penetrate easily through the cell membrane in the absence of endocytosis, while some endocytosis is observed for larger particle clusters. Once inside, the particles are sequestered in vesicles, which continue to fill up with increasing incubation time till they rupture. Particles coated with a dense grafted polymer brush are also tested, and, using flow cytometry, are shown to prevent adherence to the cell membrane and hence penetration of the cell, which effectively decreases reactive oxygen species (ROS) formation and protects cells, even in the absence of light exposure. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating can potentially play an important role in protecting cells and tissue from damage. PMID:19197964

  17. Effects of silver nanoparticles on human dermal fibroblasts and epidermal keratinocytes.

    PubMed

    Galandáková, A; Franková, J; Ambrožová, N; Habartová, K; Pivodová, V; Zálešák, B; Šafářová, K; Smékalová, M; Ulrichová, J

    2016-09-01

    Biomedical application of silver nanoparticles (AgNPs) has been rapidly increasing. Owing to their strong antimicrobial activity, AgNPs are used in dermatology in the treatment of wounds and burns. However, recent evidence for their cytotoxicity gives rise to safety concerns. This study was undertaken as a part of an ongoing programme in our laboratory to develop a topical agent for wound healing. Here, we investigated the potential toxicity of AgNPs using normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) with the aim of comparing the effects of AgNPs and ionic silver (Ag-I). Besides the effect of AgNPs and Ag-I on cell viability, the inflammatory response and DNA damage in AgNPs and Ag-I-treated cells were examined. The results showed that Ag-I were significantly more toxic than AgNPs both on NHDF and NHEK. Non-cytotoxic concentrations of AgNPs and Ag-I did not induce DNA strand breaks and did not affect inflammatory markers, except for a transient increase in interleukin 6 levels in Ag-I-treated NHDF. The results showed that AgNPs are more suitable for the intended application as a topical agent for wound healing up to the concentration 25 µg/mL. PMID:26500221

  18. Enhanced effect of fibroblast growth factor-2-containing dalteparin/protamine nanoparticles on hair growth

    PubMed Central

    Takabayashi, Yuki; Nambu, Masaki; Ishihara, Masayuki; Kuwabara, Masahiro; Fukuda, Koichi; Nakamura, Shingo; Hattori, Hidemi; Kiyosawa, Tomoharu

    2016-01-01

    Purpose Although treatments for alopecia are in high demand, not all treatments are safe and reliable. Dalteparin/protamine nanoparticles (D/P NPs) can effectively carry growth factors (GFs) such as fibroblast GF (FGF)-2. The purpose of this study was to identify the effects of FGF-2-containing D/P NPs (FGF-2&D/P NPs) on hair growth. Patients and methods In this study, the participants were 12 volunteers with thin hair. One milliliter of FGF-2 (100 ng/mL) and D/P NPs (56 μg/mL) was applied and massaged on the skin of the scalp by the participants twice a day. They were evaluated for 6 months. Participants were photographed using a digital camera for general observation and a hair diagnosis system for measuring hair diameter. Results The mean diameter of the hairs was significantly higher following the application of FGF-2&D/P NPs for 6 months. Objective improvements in thin hair were observed in two cases. Nine participants experienced greater bounce and hair resilience. Conclusion The transdermal application of FGF-2&D/P NPs to the scalp can be used as a new treatment for alopecia. PMID:27274299

  19. Combined treatment with vitamin E and gefitinib has synergistic effects to inhibit TGF-β1-induced renal fibroblast proliferation.

    PubMed

    Yiang, Giou-Teng; Chen, Jen-Ni; Lin, Pei-Shiuan; Liu, Hsiao-Chun; Chen, Shu-Ying; Wei, Chyou-Wei

    2016-06-01

    Renal fibroblast proliferation is key in renal fibrosis and chronic kidney disease. Transforming growth factor-β1 (TGF-β1) has been demonstrated to be an important factor that induces cell proliferation in renal fibroblasts. Epidermal growth factor receptor (EGFR) is also recognized as a factor promoting renal fibroblast proliferation. In addition, mitogen‑activated protein kinase signaling pathways are associated with TGF‑β1‑ and EGFR‑induced cell proliferation. Gefitinib, an EGFR tyrosine kinase inhibitor, is predominantly used as an anti‑tumor therapeutic agent in clinical therapeutic strategies. However, gefitinib has been suggested to exert anti‑proliferative effects on renal fibroblasts, however, high‑dose gefitinib may result in serious side effects. The present study aims to determine whether low‑dose gefitinib reduces gefitinib‑induced side effects and maintains the anti‑proliferative effects on renal fibroblasts. TGF‑β1 promotes cell proliferation in renal fibroblasts, and the current study demonstrates that low‑dose gefitinib treatment exhibits anti‑proliferative effects similar to those of high‑dose gefitinib treatment. Thus, although high‑dose gefitinib is a conventional anti‑tumor drug, low‑dose gefitinib may be of use in renal fibrosis treatment. Furthermore, the present study demonstrates that a combined treatment with low-dose gefitinib and vitamin E has synergistic effects that reduce TGF‑β1‑induced fibroblast proliferation, cell-cycle arrest and the ERK phosphorylation pathway. PMID:27109695

  20. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: ensuing energetic and oxidative stress implications.

    PubMed

    Pardo-Andreu, Gilberto L; Nuñez-Figueredo, Yanier; Tudella, Valeria G; Cuesta-Rubio, Osmany; Rodrigues, Fernando P; Pestana, Cezar R; Uyemura, Sérgio A; Leopoldino, Andreia M; Alberici, Luciane C; Curti, Carlos

    2011-06-15

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²⁺ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. PMID:21549140

  1. Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery.

    PubMed

    Clelland, Lyndsay J; Browne, Brendan M; Alvarez, Silvina M; Miner, Amy S; Ratz, Paul H

    2011-09-01

    K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 μM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in β-escin permeabilized tissue. GF-109203X at 1 μM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 μM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and β-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that β-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization. PMID:21706258

  2. The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

    PubMed

    Wetzels, A M; Punt-Van der Zalm, A P; Bastiaans, B A; Janssen, B A; Goverde, H J; Rolland, R

    1992-07-01

    A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified. PMID:1500485

  3. Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis

    PubMed Central

    Bianchessi, Marta; Chen, Yuwen; Durgam, Sushmitha; Pondenis, Holly; Stewart, Matthew

    2016-01-01

    Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity. PMID:26839571

  4. Simulated studies on the biological effects of space radiation on quiescent human fibroblasts

    NASA Astrophysics Data System (ADS)

    Ding, Nan; Pei, Hailong; He, Jinpeng; Furusawa, Yoshiya; Hirayama, Ryoichi; Liu, Cuihua; Matsumoto, Yoshitaka; Li, He; Hu, Wentao; Li, Yinghui; Wang, Jufang; Wang, Tieshan; Zhou, Guangming

    2013-10-01

    High charge and energy (HZE) particles are severe risk to manned long-term outer space exploration. Studies on the biological effects of space HZE particles and the underlying mechanisms are essential to the accurate risk assessment and the development of efficient countermeasure. Since majority of the cells in human body stay quiescent (G0 phase), in this study, we established G0 cell and G1 cell models by releasing human normal embryonic lung fibroblast cells from contact inhibition and studied the radiation toxicity of various kinds of HZE particles. Results showed that all of the particles were dose-dependently lethal and G0 cells were more radioresistant than G1 cells. We also found that 53BP1 foci were induced in a LET- and fluence-dependent manner and fewer foci were induced in G0 cells than G1 cells, however, the decrease of foci in 24 h after irradiation was highly relevant to the type of particles. These results imply that even though health risk of space radiation is probably overestimated by the data obtained with exponentially growing cells, whose radiosensitivity is similar to G1 cells, the risk of space HZE particles is un-ignorable and accurate assessment and mechanistic studies should be deepened. The diverse abilities of G0 cells and G1 cells in repairing DNA damages induced by HZE particles emphasize the importance in studying the impact of HZE particles on DNA damage repair pathways.

  5. Protective effect of porphyra-334 on UVA-induced photoaging in human skin fibroblasts.

    PubMed

    Ryu, Jina; Park, Su-Jin; Kim, In-Hye; Choi, Youn Hee; Nam, Taek-Jeong

    2014-09-01

    The significant increase in life expectancy is closely related to the growing interest in the impact of aging on the function and appearance of the skin. Skin aging is influenced by several factors, and solar ultraviolet (UV) irradiation is considered one of the most important causes of skin photoaging. The aim of this study was to examine the anti-photoaging role of porphyra-334 from Porphyra (P.) yezoensis, a mycosporine-like amino acid (MAA), using high-performance liquid chromatography (HPLC), and electrospray ionization‑mass spectrometry (ESI-MS). In the present study, extracted UV‑absorbing compounds from P. yezoensis included palythine, asterina-330 and porphyra-334. Porphyra-334 was the most abundant MAA in P. yezoensis, and it was therefore used for conducting antiphotoaging experiments. The effect of porphyra-334 on the prevention of photoaging was investigated by measuring reactive oxygen species (ROS) production and matrix metalloproteinase (MMP) levels, as well as extracellular matrix (ECM) components and protein expression in UVA‑irradiated human skin fibroblasts. Porphyra-334 suppressed ROS production and the expression of MMPs following UVA irradiation, while increasing levels of ECM components, such as procollagen, type I collagen, elastin. These results suggest that porphyra-334 has various applications in cosmetics and toiletries because of its anti‑photoaging activities and may serve as a novel anti-aging agent. PMID:24946848

  6. Effects of NOX1 on fibroblastic changes of endothelial cells in radiation‑induced pulmonary fibrosis.

    PubMed

    Choi, Seo-Hyun; Kim, Miseon; Lee, Hae-June; Kim, Eun-Ho; Kim, Chun-Ho; Lee, Yoon-Jin

    2016-05-01

    Lung fibrosis is a major complication in radiation‑induced lung damage following thoracic radiotherapy, while the underlying mechanism has remained to be elucidated. The present study performed immunofluorescence and immunoblot assays on irradiated human pulmonary artery endothelial cells (HPAECs) with or without pre‑treatment with VAS2870, a novel NADPH oxidase (NOX) inhibitor, or small hairpin (sh)RNA against NOX1, ‑2 or ‑4. VAS2870 reduced the cellular reactive oxygen species content induced by 5 Gy radiation in HPAECs and inhibited phenotypic changes in fibrotic cells, including increased alpha smooth muscle actin and vimentin, and decreased CD31 and vascular endothelial cadherin expression. These fibrotic changes were significantly inhibited by treatment with NOX1 shRNA, but not by NOX2 or NOX4 shRNA. Next, the role of NOX1 in pulmonary fibrosis development was assessed in the lung tissues of C57BL/6J mice following thoracic irradiation using trichrome staining. Administration of an NOX1‑specific inhibitor suppressed radiation‑induced collagen deposition and fibroblastic changes in the endothelial cells (ECs) of these mice. The results suggested that radiation‑induced pulmonary fibrosis may be efficiently reduced by specific inhibition of NOX1, an effect mediated by reduction of fibrotic changes of ECs. PMID:27053172

  7. Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts

    PubMed Central

    Liu, Xiangning; Zhou, Xiaosong; Li, Shaobing; Lai, Renfa; Zhou, Zhiying; Zhang, Ye; Zhou, Lei

    2014-01-01

    Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid. PMID:24623977

  8. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-09-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

  9. Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts

    PubMed Central

    Alili, Lirija; Chapiro, Swetlana; Marten, Gernot U.; Schmidt, Annette M.; Zanger, Klaus; Brenneisen, Peter

    2015-01-01

    Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles. PMID:26090418

  10. Effects of NOX1 on fibroblastic changes of endothelial cells in radiation-induced pulmonary fibrosis

    PubMed Central

    CHOI, SEO-HYUN; KIM, MISEON; LEE, HAE-JUNE; KIM, EUN-HO; KIM, CHUN-HO; LEE, YOON-JIN

    2016-01-01

    Lung fibrosis is a major complication in radiation-induced lung damage following thoracic radiotherapy, while the underlying mechanism has remained to be elucidated. The present study performed immunofluorescence and immunoblot assays on irradiated human pulmonary artery endothelial cells (HPAECs) with or without pre-treatment with VAS2870, a novel NADPH oxidase (NOX) inhibitor, or small hairpin (sh)RNA against NOX1, -2 or -4. VAS2870 reduced the cellular reactive oxygen species content induced by 5 Gy radiation in HPAECs and inhibited phenotypic changes in fibrotic cells, including increased alpha smooth muscle actin and vimentin, and decreased CD31 and vascular endothelial cadherin expression. These fibrotic changes were significantly inhibited by treatment with NOX1 shRNA, but not by NOX2 or NOX4 shRNA. Next, the role of NOX1 in pulmonary fibrosis development was assessed in the lung tissues of C57BL/6J mice following thoracic irradiation using trichrome staining. Administration of an NOX1-specific inhibitor suppressed radiation-induced collagen deposition and fibroblastic changes in the endothelial cells (ECs) of these mice. The results suggested that radiation-induced pulmonary fibrosis may be efficiently reduced by specific inhibition of NOX1, an effect mediated by reduction of fibrotic changes of ECs. PMID:27053172

  11. Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

    PubMed

    Viteri, Gabriela; Chung, Youn Wook; Stadtman, Earl R

    2010-01-01

    The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging. PMID:19958786

  12. Fibroproliferative effect of microRNA-21 in hypertrophic scar derived fibroblasts.

    PubMed

    Li, Guangzao; Zhou, Renpeng; Zhang, Qi; Jiang, Banghong; Wu, Qingkai; Wang, Chen

    2016-07-01

    Hypertrophic scar (HS) is a fibroproliferative disorder caused by abnormal wound healing, which is characterized by excessive deposition of extracellular matrix (ECM) secreted by fibroblasts. We previous have found that expression of microRNA-21(miR-21) was increased in tissues and fibroblasts of HS. However, the underlying molecular mechanism remains to be further elucidated. In this study, we identified the miR-21 was a marker for the phenotype of HS fibroblasts, as anti-miR-21 reduced expression of fibrosis markers such as Col1A1, Col3A1, Fn and α-SMA in fibroblasts and overexpression of miR-21 promoted fibroproliferative expression in fibroblasts. Furthermore, we also found that miR-21 promoted TGF-β1 induced fibroproliferative expression by repressing Smad7 expression in vitro. In addition, the miR-21 inhibitor inhibited the growth of hypertrophic scar tissue in vivo (nude mice experimental model). These results indicated that miR-21 was a critical regulator for HS formation and TGF- β1/miR-21/Smad7 pathway could be a useful therapeutic target for the treatment of HS. PMID:27207585

  13. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Astrophysics Data System (ADS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-10-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/μm to 975 KeV/gmm with particle energy (on the cells) between 94 - 603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/μm. The inactivation cross-section (αi) and the action-section for mutant induction (αm) ranged from 2.2 to 92.0 μm2 and 0.09 to 5.56 × 10-3 μm2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/μm. The mutagenicity (αm/αi) ranged from 2.05 to 7.99 × 10-5 with the maximum value at 150 keV/μm. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  14. Metabolomic Elucidation of the Effects of Curcumin on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis.

    PubMed

    Ahn, Joong Kyong; Kim, Sooah; Hwang, Jiwon; Kim, Jungyeon; Lee, You Sun; Koh, Eun-Mi; Kim, Kyoung Heon; Cha, Hoon-Suk

    2015-01-01

    Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and joint disability. Curcumin is known to be effective in ameliorating joint inflammation in RA. To obtain new insights into the effect of curcumin on primary fibroblast-like synoviocytes (FLS, N = 3), which are key effector cells in RA, we employed gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS)-based metabolomics. Metabolomic profiling of tumor necrosis factor (TNF)-α-stimulated and curcumin-treated FLS was performed using GC/TOF-MS in conjunction with univariate and multivariate statistical analyses. A total of 119 metabolites were identified. Metabolomic analysis revealed that metabolite profiles were clearly distinct between TNF-α-stimulated vs. the control group (not stimulated by TNF-α or curcumin). Treatment of FLS with curcumin showed that the metabolic perturbation by TNF-α could be reversed to that of the control group to a considerable extent. Curcumin-treated FLS had higher restoration of amino acid and fatty acid metabolism, as indicated by the prominent metabolic restoration of intermediates of amino acid and fatty acid metabolism, compared with that observed in TNF-α-stimulated FLS. In particular, the abundance of glycine, citrulline, arachidonic acid, and saturated fatty acids in TNF-α-stimulated FLS was restored to the control level after treatment with curcumin, suggesting that the effect of curcumin on preventing joint inflammation may be elucidated with the levels of these metabolites. Our results suggest that GC/TOF-MS-based metabolomic investigation using FLS has the potential for discovering the mechanism of action of curcumin and new targets for therapeutic drugs in RA. PMID:26716989

  15. Effect of advanced glycation endproducts on gene expression profiles of human dermal fibroblasts.

    PubMed

    Molinari, J; Ruszova, E; Velebny, V; Robert, L

    2008-06-01

    The Maillard reaction and its end products, AGE-s (Advanced Glycation End products) are rightly considered as one of the important mechanisms of post-translational tissue modifications with aging. We studied the effect of two AGE-products prepared by the glycation of lysozyme and of BSA, on the expression profile of a large number of genes potentially involved in the above mentioned effects of AGE-s. The two AGE-products were added to human skin fibroblasts and gene expression profiles investigated using microarrays. Among the large number of genes monitored the expression of 16 genes was modified by each AGE-preparations, half of them only by both of them. Out of these 16 genes, 12 were more strongly affected, again not all the same for both preparations. Both of them upregulated MMP and serpin-expression and downregulated some of the collagen-chain coding genes, as well as the cadherin- and fibronectin genes. The BSA-AGE preparation downregulated 10 of the 12 genes strongly affected, only the serpin-1 and MMP-9 genes were upregulated. The lysozyme-AGE preparation upregulated selectively the genes coding for acid phosphatase (ACP), integrin chain alpha5 (ITGA5) and thrombospondin (THBS) which were unaffected by the BSA-AGE preparation. It was shown previously that the lysozyme-AGE strongly increased the rate of proliferation and also cell death, much more than the BSA-AGE preparation. These differences between these two AGE-preparations tested suggest the possibility of different receptor-mediated transmission pathways activated by these two preparations. Most of the gene-expression modifications are in agreement with biological effects of Maillard products, especially interference with normal tissue structure and increased tissue destruction. PMID:18297408

  16. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  17. Metabolomic Elucidation of the Effects of Curcumin on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

    PubMed Central

    Hwang, Jiwon; Kim, Jungyeon; Lee, You Sun; Koh, Eun-Mi; Kim, Kyoung Heon; Cha, Hoon-Suk

    2015-01-01

    Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and joint disability. Curcumin is known to be effective in ameliorating joint inflammation in RA. To obtain new insights into the effect of curcumin on primary fibroblast-like synoviocytes (FLS, N = 3), which are key effector cells in RA, we employed gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS)-based metabolomics. Metabolomic profiling of tumor necrosis factor (TNF)-α-stimulated and curcumin-treated FLS was performed using GC/TOF-MS in conjunction with univariate and multivariate statistical analyses. A total of 119 metabolites were identified. Metabolomic analysis revealed that metabolite profiles were clearly distinct between TNF-α-stimulated vs. the control group (not stimulated by TNF-α or curcumin). Treatment of FLS with curcumin showed that the metabolic perturbation by TNF-α could be reversed to that of the control group to a considerable extent. Curcumin-treated FLS had higher restoration of amino acid and fatty acid metabolism, as indicated by the prominent metabolic restoration of intermediates of amino acid and fatty acid metabolism, compared with that observed in TNF-α-stimulated FLS. In particular, the abundance of glycine, citrulline, arachidonic acid, and saturated fatty acids in TNF-α-stimulated FLS was restored to the control level after treatment with curcumin, suggesting that the effect of curcumin on preventing joint inflammation may be elucidated with the levels of these metabolites. Our results suggest that GC/TOF-MS-based metabolomic investigation using FLS has the potential for discovering the mechanism of action of curcumin and new targets for therapeutic drugs in RA. PMID:26716989

  18. Antigenotoxic and antimutagenic effects of diphenyl ditelluride against several known mutagens in Chinese hamster lung fibroblasts.

    PubMed

    Trindade, Cristiano; Juchem, André L M; de Albuquerque, Nathália R M; de Oliveira, Iuri M; Rosa, Renato M; Guecheva, Temenouga N; Saffi, Jenifer; Henriques, João A P

    2015-11-01

    The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 μM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 μM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 μM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 μM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 μM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells. PMID:26001756

  19. Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  20. Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

    NASA Astrophysics Data System (ADS)

    Basso, F. G.; Pansani, T. N.; Turrioni, A. P. S.; Kurachi, C.; Bagnato, V. S.; Hebling, J.; de Souza Costa, C. A.

    2013-05-01

    Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions.

  1. The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

    SciTech Connect

    Ndukwe Erlingsson, Uzochi Chimdinma; Iacobazzi, Francesco; Liu, Aiping; Ardon, Orly; Pasquali, Marzia; Longo, Nicola

    2013-08-09

    Highlights: •Valinomycin can cause mitochondrial stress and stimulate fatty acid oxidation. •Cells with VLCAD deficiency fail to increase fatty acid oxidation in response to valinomycin. •Response to valinomycin can help in the diagnosis of VLCAD deficiency. -- Abstract: Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP{sup +}) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP{sup +} accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [{sup 14}C]-palmitate oxidation (measured as [{sup 14}C]O{sub 2} release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.

  2. Effect of wavelength and fluence on morphology, cellular and genetic integrity of diabetic wounded human skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Abrahamse, H.; Hawkins, D.; Houreld, N.

    2006-02-01

    An alternative treatment modality for diabetic wound healing includes low level laser therapy (LLLT). Biostimulation of such wounds may be of benefit to patients by reducing healing time. Structural, cellular and genetic events in diabetic wounded human skin fibroblasts (WS1) were evaluated after exposing cells in culture to a Helium-Neon (632.8nm), a Diode laser (830nm) and a Nd:YAG (Neodynium:Yttrium-Allumina-Gallium) laser (1064nm) at either 5J/cm2 or 16J/cm2. Cells were exposed twice a week and left 24 hours post-irradiation prior to measuring effects. Structural changes were evaluated by assessing colony formation, haptotaxis and chemotaxis. Cellular changes were evaluated using cell viability, (adenosine-triphosphate, ATP production), and proliferation, (alkaline phosphatase, ALP and basic fibroblast growth factor, bFGF expression), while the Comet assay evaluated DNA damage and cytotoxicity was determined assessing membrane permeability for lactate dehydrogenase (LDH). Caspase 3/7 activity was used as an estimate of apoptosis as a result of irradiation. The irradiated diabetic wounded cells showed structural, cellular as well as molecular resilience comparable to that of unwounded normal skin fibroblast cells. With regards to fluence, 5J/cm2 elicit positive cellular and structural responses while 16J/cm2 increases cellular and genetic damage and cellular morphology is altered. Different wavelengths of LLLT influences the beneficial outcomes of diabetic wounded cells and although all three wavelengths elicit cellular effects, the penetration depth of 830nm plays a significant role in the healing of diabetic wounded human fibroblast cells. Results from this study validate the contribution of LLLT to wound healing and elucidate the biochemical effects at a cellular level while highlighting the role of different dosages and wavelengths in LLLT.

  3. Efficient delivery to human lung fibroblasts (WI-38) of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor and its inhibitory effect on collagen synthesis in idiopathic pulmonary fibrosis.

    PubMed

    Togami, Kohei; Miyao, Aki; Miyakoshi, Kei; Kanehira, Yukimune; Tada, Hitoshi; Chono, Sumio

    2015-01-01

    In the present in vitro study, we assessed the delivery of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor (tbFGF) to lung fibroblasts and investigated the anti-fibrotic effect of the drug. The tbFGF peptide, KRTGQYKLC, was used to modify the surface of liposomes (tbFGF-liposomes). We used the thin-layer evaporation method, followed by sonication, to prepare tbFGF-liposomes containing pirfenidone. The cellular accumulation of tbFGF-liposomes was 1.7-fold greater than that of non-modified liposomes in WI-38 cells used as a model of lung fibroblasts. Confocal laser scanning microscopy showed that tbFGF-liposomes were widely localized in WI-38 cells. The inhibitory effects of pirfenidone incorporated into tbFGF-liposomes on transforming growth factor-β1 (TGF-β1)-induced collagen synthesis in WI-38 cells were evaluated by measuring the level of intracellular hydroxyproline, a major component of the protein collagen. Pirfenidone incorporated into tbFGF-liposomes at concentrations of 10, 30, and 100 µM significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells. The anti-fibrotic effect of pirfenidone incorporated into tbFGF-liposomes was enhanced compared with that of pirfenidone solution. These results indicate that tbFGF-liposomes are a useful drug delivery system of anti-fibrotic drugs to lung fibroblasts for the treatment of idiopathic pulmonary fibrosis. PMID:25747986

  4. Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro.

    PubMed

    Clark, W A

    1976-09-01

    The stability of the differentiated state of cardiac myocytes in vitro was examined under culture conditions which selectively stimulated or inhibited proliferation of fibroblasts. Regulation of fibroblast proliferation in cultures of myocardial cells from 8-day embryonic chicks was achieved by adjustment of the glutamine (Gln) concentration in the culture medium (Ham's F-12 medium containing 2 x amino acids and 5% fetal calf serum). Myocardial cells, when plated at 80 cells/mm2 in Gln- medium, maintained a stable density of approximately 40% of the plating density for more than 30 days. When Gln was added to the medium (292 micrograms/ml) fibroblast proliferation was stimulated, and by 5-6 days after this addition cell densities had increased to confluency. The selective action of glutamine on fibroblast proliferation was determined by labeling cultures with tritiated thymidine ([3H]TdR) and scoring its incorporation into myocytes and fibroblasts by radioautography. After 2 weeks in Gln- medium, the mitotic index was 0.3% and the [3H]TdR-labeling index (1.5-hr pulse) was 6.4%. In addition, the proportion of myocytes in the population was constant at 64.2% for at least 30 days in vitro, and contractile activity was observed for up to 6 months. After 5 days of Gln replacement, the cells exhibited a labeling index of 25%, the proportion of myocytes decreased to less than 10% and contractile activity was rarely observed. Although the [3H]TdR-labeling index of fibroblasts and myocytes was nearly identical in Gln- medium, the addition of Gln produced a fivefold stimulation in the fibroblast labeling index, but did not affect myocyte proliferation or DNA synthesis. A unique phenomenon of myocyte congregation was observed only in Gln- medium which resulted in the formation of myocyte colonies from which fibroblasts were largely absent. It is suggested that this process with the resultant establishment of a functional electrical syncytium plays a significant role in the

  5. Effects of ultraviolet radiation on intercellular communication in V79 Chinese hamster fibroblasts.

    PubMed

    Bånrud, H; Mikalsen, S O; Berg, K; Moan, J

    1994-02-01

    The effects of ultraviolet (UV) radiation on gap junctional intercellular communication (GJIC) in V79 Chinese hamster fibroblasts were studied by means of a dye transfer assay. Intercellular communication was shown to be altered by UVB (297/302 nm) and UVA (365 nm) radiation, the effect depending on the wavelength of exposure and time between irradiation and microinjection of the dye in the dye transfer assay. Exposure to 297/302 nm radiation induced a reduction in intercellular communication 6 min after exposure. Incubation of the cells post-irradiation reversed the inhibition of GJIC. From 2 to 24 h after exposure an increase in GJIC over the control cells was seen, with a maximum at 8 h post-irradiation. UVA (365 nm) radiation, on the other hand, induced an increase in the intercellular communication 6 min after irradiation. Incubation of the cells post-irradiation led to a decrease in the number of communicating cells, with a minimum seen 4 h after exposure. The reduction in communication observed after exposure to UVB and UVA was not correlated with similar modifications in the gap junction protein connexin43 as found when exposing the cells to the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. For the higher fluences of UVA, a decrease in immunorecognizable connexin43 was seen, concomitant with a markedly increased background of higher mol. wt compounds. This may be due to UVA-induced crosslinking of connexin43. No correlation was found between changes in communication induced by UV radiation and levels of cyclic AMP. PMID:8313514

  6. Cryoprotective effect of low-molecular-weight hyaluronan on human dermal fibroblast monolayers.

    PubMed

    Ujihira, Masanobu; Iwama, Akira; Aoki, Makie; Aoki, Kanako; Omaki, Sayaka; Goto, Erika; Mabuchi, Kiyoshi

    2010-01-01

    The purpose of this study was to assess the availability of low-molecular-weight (low-MW) hyaluronan (HA) as a cryoprotectant for cellular cryopreservation. To clarify whether low-MW HA is cryoprotective, we evaluated the effect of HA concentration (0-5% w/w) in a cryoprotectant solution on cell membrane integrity after freeze-thaw. A test sample was created using human dermal fibroblast monolayers incubated in a culture dish for 24 h (37 degrees C, 5% CO2). Sodium hyaluronate (MW 3 x 10(4)-5 x 10(4)) dissolved in medium served as the cryoprotectant solution. Samples were immersed in the solution for 2 h at 0-4 degrees C. They were frozen at a cooling rate of 3 degrees C/min from 4 to -80 degrees C, cooled further to below -185 degrees C, and then thawed. Cell membrane integrity after thawing was evaluated using a trypan blue exclusion assay. The sample and freezing procedures were repeated in subsequent experiments, while the conditions of the solution immersion with respect to the sample varied. Next, to clarify whether the cryoprotective action of HA is intra- or extracellular, we performed three experiments. The first studied the dependence of membrane integrity after freeze-thaw on preliminary incubation time (0.75-24 h at 37 degrees C) with a sample immersed in the solution (5% w/w HA). In the second, membrane integrity of thawed samples that were initially frozen in a medium instead of solution, by removing extracellular HA following a preliminary 6-h incubation period, were evaluated. Thirdly, we investigated cellular uptake of fluorescein isothiocyanate-labeled HA (MW 10(5), 1% w/w) after a preliminary 6-h incubation period under fluorescent microscopy (without freeze-thaw). The results show that HA had a cryoprotective effect, and that this cryoprotective action was intracellular. Therefore, low- MW HA proves to be a promising cellular cryoprotectant. PMID:20687452

  7. [Effect of elastin peptides on the production of matrix metalloproteinase 2 by human skin fibroblasts in culture].

    PubMed

    Huet, E; Brassart, B; Wallach, J; Debelle, L; Haye, B; Emonard, H; Hornebeck, W

    2001-01-01

    Soluble elastin-derived peptides from alkaline or elastase hydrolysis of insoluble elastin, as well as tropoelastin, increase matrix metalloproteinase-2 (MMP-2) production by human skin fibroblasts in culture as determined by gelatin zymography and ELISA. Such an effect is time and concentration dependent; it can be reproduced by synthetic elastin: VGVAPG, PGAIPG, and laminin: LGTIPG, hexapeptides and inhibited by lactose and is therefore elastin receptor-mediated. The steady state levels of MMP-2 mRNAs are invariant following elastin-fibroblasts interaction. Inhibition of phospholipase C (D-609), ADP-ribosylation factor (brefeldin), protein kinase C (RO-318220) and phospholipase D (1-propanol) totally abolished the elastin-mediated increase of MMP-2 production. It suggested that the post-transcriptional mechanism controlling the elastin-mediated overproduction of MMP-2 involved a cascade leading to phospholipase D activation. PMID:11723829

  8. Effects of Dietary Phosphate and Calcium Intake on Fibroblast Growth Factor-23

    PubMed Central

    van Ittersum, Frans J.; Büttler, Rahel M.; Heijboer, Annemieke C.; Blankenstein, Marinus A.; ter Wee, Piet M.

    2011-01-01

    Summary Background and objectives Little is known about the influence of dietary phosphate intake on fibroblast growth factor-23 (FGF23) and its subsequent effects on vitamin D levels. This study addresses changes in intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23), phosphaturia, and levels of vitamin D on high and low phosphate and calcium intake. Design, setting, participants, & measurements Ten healthy subjects adhered to a diet low or high in phosphate and calcium content for 36 hours each with a 1-week interval during which subjects adhered to their usual diet. Serum phosphate, calcium, vitamin D metabolites, parathyroid hormone (PTH), and FGF23 levels (cFGF23 and iFGF23) were measured several times a day. Phosphate, calcium, and creatinine excretion was measured in 24-hour urine on all study days. Results Serum phosphate levels and urinary phosphate increased during high dietary phosphate intake (from 1.11 to 1.32 mmol/L, P < 0.0001 and 21.6 to 28.8 mmol/d, P = 0.0005, respectively). FGF23 serum levels increased during high dietary phosphate/calcium intake (cFGF23 from 60 to 72 RU/ml, P < 0.001; iFGF23 from 33 to 37 ng/L, P = 0.003), whereas PTH declined. 1,25-Dihydroxyvitamin D (1,25D) showed an inverse relation with FGF23. Conclusions Variation in dietary phosphate and calcium intake induces changes in FGF23 (on top of a circadian rhythm) and 1,25D blood levels as well as in urinary phosphate excretion. These changes are detectable the day after the change in the phosphate content of meals. Higher FGF23 levels are associated with phosphaturia and a decline in 1,25D levels. PMID:21030580

  9. Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts.

    PubMed

    Tokudome, Yoshihiro; Nakamura, Kyosuke; Kage, Madoka; Todo, Hiroaki; Sugibayashi, Kenji; Hashimoto, Fumie

    2012-09-01

    The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together. PMID:22264122

  10. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    PubMed Central

    2013-01-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

  11. GENOTOXIC EFFECTS OF COMPLEX MARINE SEDIMENT EXTRACTS ON V79 CHINESE HAMSTER LUNG FIBROBLASTS

    EPA Science Inventory

    A mammalian in vitro system was used to evaluate the genotoxic potential of two complex environmental samples. ister chromatid exchanges (SCEs) were measured in Chinese hamster V79 lung fibroblast cells, following exposure to whole extracts of sediments collected from a highly co...

  12. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: Specific effect of transforming growth factor. beta

    SciTech Connect

    Eghbali, M.; Tomek, R.; Woods, C.; Bhambi, B. )

    1991-02-01

    Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-{beta}{sub 1}, (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-{beta}{sub 1} became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-{beta}{sub 1}, cell proliferation (as measured by ({sup 3}H)thymidine incorporation) was moderately increased. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-{beta}{sub 1} in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. The findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-{beta}{sub 1} seems to be a specific inducer of such conversion.

  13. The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts.

    PubMed

    Lu, Jian; Wei, Caihong; Zhang, Xiaoning; Xu, Lingyang; Zhang, Shifang; Liu, Jiasen; Cao, Jiaxue; Zhao, Fuping; Zhang, Li; Li, Bichun; Du, Lixin

    2013-06-01

    Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may promote muscle growth, we used RNA interference mediated by a lentiviral vector to knockdown myostatin in goat fetal fibroblast cells. We also investigated the expression changes in relevant myogenic regulatory factors (MRFs) and adipogenic regulatory factors in the absence of myostatin in goat fetal fibroblasts. Quantitative RT-PCR revealed that myostatin transcripts were significantly reduced by 75 % (P < 0.01). Western blot showed that myostatin protein expression was reduced by 95 % (P < 0.01). We also found that the mRNA expression of activin receptor IIB (ACVR2B) significantly increased by 350 % (P < 0.01), and p21 increased 172 % (P < 0.01). Furthermore, myostatin inhibition decreased Myf5 and increased MEF2C mRNA expression in goat fetal fibroblasts, suggesting that myostatin regulates MRFs differently in fibroblasts compared to muscle. In addition, the expression of adipocyte marker genes peroxisome proliferator-activated receptor (PPAR) γ and leptin, but not CCAAT/enhance-binding protein (C/EBP) α and C/EBPβ, were upregulated at the transcript level after myostatin silencing. These results suggest that we have generated a novel way to block myostatin in vitro, which could be used to improve livestock meat production and gene therapy of musculoskeletal diseases. This also suggests that myostatin plays a negative role in regulating the expression of adipogenesis related genes in goat fetal fibroblasts. PMID:23604693

  14. Genome-wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

    PubMed Central

    2014-01-01

    Background Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. PMID:24485159

  15. The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation.

    PubMed

    Wetzels, A M; Bastiaans, B A; Hendriks, J C; Goverde, H J; Punt-van der Zalm, A P; Verbeet, J G; Braat, D D

    1998-05-01

    In a human in-vitro fertilization (IVF) programme, the effect of co-culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients. PMID:9647567

  16. Effect of ultrasound irradiation on α-SMA and TGF-β1 expression in human dermal fibroblasts.

    PubMed

    Maeshige, Noriaki; Terashi, Hiroto; Aoyama, Michiko; Torii, Kazuhiro; Sugimoto, Masaharu; Usami, Makoto

    2011-01-01

    Ultrasound therapy is used to promote pressure ulcer healing as an adjunctive therapy. However, the efficacy and the scientific basis of this treatment are unclear. We investigated the effect of ultrasound irradiation on alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1) expression in human dermal fibroblasts. These are important factors for acceleration of wound closure. We used pulsed ultrasound of 0, 0.1, 0.5, and 1.0 W/cm2. TGF-β1 and α-SMA mRNA was measured by quantitative real-time polymerase chain reaction, α-SMA protein was examined by western blot, and localization of α-SMA was evaluated by immunofluorescence staining. Expression of α-SMA and TGF-β1 mRNA was increased at 24 h but not at 48 h after ultrasound irradiation. There were significant differences between controls of 0 W/cm² and 0.1 W/cm² with a 1.34 ± 0.26 fold increase in α-SMA (P < 0.05) and a 1.78 ± 0.57 fold increase in TGF-β1 (P < 0.05). Protein levels of α-SMA were also increased and detected in ultrasound irradiated fibroblasts at 24 h. Ultrasound irradiation promotes α-SMA expression in human dermal fibroblasts and this suggests the biological mechanism of ultrasound efficacy on chronic wound treatment. PMID:21937873

  17. Aging changes agonist induced contractile responses in permeabilized rat bladder.

    PubMed

    Durlu-Kandilci, N Tugba; Denizalti, Merve; Sahin-Erdemli, Inci

    2015-08-01

    Aging alters bladder functions where a decrease in filling, storage and emptying is observed. These changes cause urinary incontinence, especially in women. The aim of this study is to examine how aging affects the intracellular calcium movements due to agonist-induced contractions in permeabilized female rat bladder. Urinary bladder isolated from young and old female Sprague-Dawley rats were used. Small detrusor strips were permeabilized with β-escin. The contractile responses induced with agonists were compared between young and old groups. Carbachol-induced contractions were decreased in permeabilized detrusor from old rats compared to young group. Heparin and ryanodine decreased carbachol-induced contractions in young rats where only heparin inhibited these contractions in olds. Caffeine-induced contractions but not inositol triphosphate (IP3)-induced contractions were decreased in old group compared to youngs. The cumulative calcium response curves (pCa 8-4) were also decreased in old rats. Carbachol-induced calcium sensitization responses did not alter by age where GTP-β-S and GF-109203X but not Y-27632 inhibited these responses. Carbachol-induced contractions decrease with aging in rat bladder detrusor. It can be postulated as IP3-induced calcium release (IICR) is primarily responsible for the contractions in older rats where the decrease in carbachol contractions in aging may be as a result of a decrease in calcium-induced calcium release (CICR), rather than carbachol-induced calcium sensitization. PMID:26153091

  18. Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

    PubMed Central

    Hughey, Curtis C.; Hittel, Dustin S.; Johnsen, Virginia L.; Shearer, Jane

    2011-01-01

    Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis. The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples. PMID:21403632

  19. Effect of temperature and storage media on human periodontal ligament fibroblast viability.

    PubMed

    Souza, Beatriz Dulcineia Mendes; Lückemeyer, Débora Denardin; Felippe, Wilson Tadeu; Simões, Cláudia Maria Oliveira; Felippe, Mara Cristina Santos

    2010-06-01

    Many solutions have been examined as possible storage media for avulsed teeth. The purpose of this study was to compare the effectiveness of several storage media to preserve cultured periodontal ligament fibroblasts (PDLF) under different temperatures. The media tested were: sterile Hank's balanced salt solution (sHBSS), non-sterile HBSS (nHBSS), skimmed milk, Save-A-Tooth((R)), Minimum Essential Medium (MEM) and water (negative control). MEM at 37 degrees C was used as positive control. PDLF were obtained from explants of extracted healthy human teeth. Plates containing confluent PDLF were soaked in the various media for 3, 6, 24, 48 and 72 h at 37 degrees C and 20 degrees C. After incubation, viability of the cells was determined using the tetrazolium salt-based colorimetric (MTT) assay and the Trypan Blue exclusion test after 6, 24, 48 and 72 h of incubation at 20 degrees C. The results were analyzed statistically using Kruskal-Wallis, Scheffé and Mann-Whitney (alpha = 5%) tests. Results from the MTT assay at 37 degrees C and 20 degrees C showed that skimmed milk was the best storage medium for up to 24 and 48 h, respectively, followed by nHBSS and sHBSS. Results from the Trypan Blue exclusion test showed that the best storage media were milk, sHBSS and nHBSS, with no statistical differences, for any time period. The Save-A-Tooth((R)) had a detrimental effect on cells after 24 h. The influence of temperature on the effectiveness of the storage media tested showed at 20 degrees C a decreasing order of efficacy as follows: milk > sHBSS and nHBSS > MEM > Save-A-Tooth((R)) > water while at 37 degrees C it was: MEM > nHBSS > milk > sHBSS > Save-A-Tooth((R)) > water. In conclusion, incubation temperature altered the effectiveness of the storage media and skimmed milk at 20 degrees C was better than HBSS in maintaining PDLF viability. PMID:20572843

  20. Selective Permeabilization of the Blood–Brain Barrier at Sites of Metastasis

    PubMed Central

    Connell, John J.; Chatain, Grégoire; Cornelissen, Bart; Vallis, Katherine A.; Hamilton, Alastair; Seymour, Len; Sibson, Nicola R.

    2013-01-01

    Background Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood–brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases. Methods BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab (111In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of 111In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal–Wallis with Dunn’s multiple comparison test. All statistical tests were two-sided. Results Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of 111In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression. Conclusions These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor

  1. miR-146b-5p mediates p16-dependent repression of IL-6 and suppresses paracrine procarcinogenic effects of breast stromal fibroblasts

    PubMed Central

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2015-01-01

    Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16INK4A protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16INK4A suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3′UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16INK4A coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible “normalization” of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value. PMID:26338965

  2. Dabigatran, a direct thrombin inhibitor, blocks differentiation of normal fibroblasts to a myofibroblast phenotype and demonstrates anti-fibrotic effects on scleroderma lung fibroblasts

    PubMed Central

    Bogatkevich, Galina S.; Ludwicka-Bradley, Anna; Silver, Richard M.

    2010-01-01

    Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor (PAR)-1 thereby inducing normal lung fibroblasts to differentiate to a myofibroblast phenotype resembling scleroderma lung myofibroblasts. Here we demonstrate that the thrombin inhibitor dabigatran inhibits in a dose-dependant manner thrombin's induction of myofibroblasts. Dabigatran inhibits thrombin-induced cell proliferation, α-smooth muscle actin (α-SMA) expression and organization, and the production of collagen and connective tissue growth factor (CTGF). Moreover, when treated with dabigatran scleroderma lung myofibroblasts produce less CTGF, α-SMA, and collagen type I. We conclude that dabigatran restrains important profibrotic events in lung fibroblasts and that this oral direct thrombin inhibitor warrants study as a potential anti-fibrotic drug for the treatment of fibrosing lung diseases, e.g. scleroderma lung disease and idiopathic pulmonary fibrosis. PMID:19877031

  3. Collagen Gel Contraction by Fibroblasts: The Role of Myosin 2 and Gravity Effects

    NASA Technical Reports Server (NTRS)

    Johnson-Wint, Barbara P.; Malouvier, Alexandre; Holton, Emily

    1996-01-01

    Several lines of evidence suggest that collagen organization by connective tissue cells is sensitive to force. For instance, in flight experiments on rats the collagen fibrils which were produced under weightlessness and which were immediately next to the tendon fibroblasts were shown to be oriented randomly around the cells while the older fibrils right next to these and which were produced under 1 G, were highly organized.

  4. Preventive effects of tamarind seed coat extract on UVA-induced alterations in human skin fibroblasts.

    PubMed

    Phetdee, Khemjira; Rakchai, Racharat; Rattanamanee, Kwanchai; Teaktong, Thanasak; Viyoch, Jarupa

    2014-01-01

    One of the most damaging actions on skin is from solar radiation, particularly from its ultraviolet (UV) component, through the formation of oxidative species. Thus, an antioxidant strategy that prevents the formation of these oxidants could form the basis of an efficacious cutaneous protectant. Many herbal materials contain antioxidant polyphenols, and this study assessed the possibility that tamarind seed coat extract could fulfill this role. An alcoholic extract of the tamarind (Tamarindus indica L.) seed coat showed stronger antioxidant activity (2,2-diphenyl-1-picrylhydrazyl inhibition, EC(50) = 12.9 μg/ml) than L-ascorbic acid (EC(50) = 22.9 μg/ml) and α-tocopherol (EC(50) = 29.3 μg/ml). In cultured fibroblasts taken from human skin, hydrogen peroxide (100-1000 μM) damaged 62-92% of the cells compared to only 35-47% when the cells were preincubated in extract (200 μg/ml) for 24 h. UVA (40 J/cm2) irradiation of human fibroblasts damaged 25% of the cells but the death rate was reduced to 10% with extract. UV irradiation increased the proportion of cells arrest in G(0)/G(1) phase (from 59% to 78%) but this was largely prevented by the extract (64%), according to flow cytometry. Intracellular total glutathione of UVA-irradiated cells pretreated with the extract increased to 10-25% compared to the non-pretreated group at 24-72 h after irradiation. Fibroblasts typically increased matrix metalloproteinase-1 secretion after photodamage, and this is prevented by the extract. This is the first report showing that tamarind seed coat extract is an antioxidant and can protect human skin fibroblasts from cellular damage produced by UVA and thus may form the foundation for an antiaging cosmetic. PMID:24602819

  5. The Effect of SHH-Gli Signaling Pathway on the Synovial Fibroblast Proliferation in Rheumatoid Arthritis.

    PubMed

    Qin, Suping; Sun, Dexu; Li, Hui; Li, Xiangyang; Pan, Wei; Yan, Chao; Tang, Renxian; Liu, Xiaomei

    2016-04-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by chronic synovitis. This study aims to investigate the role of sonic hedgehog (SHH)-Gli signaling pathway in synovial fibroblast proliferation in rheumatoid arthritis. The expression of serum SHH in RA patients group was significantly increased compared with the systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), and healthy subject (healthy control, HC) groups, respectively; serum SHH expression of RA patients was positively correlated with rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP Ab), while there was no significant correlation between SHH expression and erythrocyte sedimentation rate (ESR). SHH, Ptch, Smo, and Gli molecules were highly expressed in rat RA-synovial fibroblast (RA-SF); after blocking the SHH-Gli signaling pathway with a Gli specific inhibitor, Gli-antagonist 61 (GANT61), RA-SF proliferation was inhibited in a dose-dependent manner and the apoptosis rate of RA-SF was increased as well; the expression levels of fibroblast growth factor receptor 1 (FGFR1) and FGFR3 declined in SF cells after GANT61 treatment. Our results suggest that SHH-Gli pathway is involved in the pathogenesis of RA, and blocking SHH-Gli pathway inhibits RA-SF cell proliferation and increases cell apoptosis, which may shed light on developing new ideas for RA treatment. PMID:26552406

  6. Free bone graft reconstruction of irradiated facial tissue: Experimental effects of basic fibroblast growth factor stimulation

    SciTech Connect

    Eppley, B.L.; Connolly, D.T.; Winkelmann, T.; Sadove, A.M.; Heuvelman, D.; Feder, J. )

    1991-07-01

    A study was undertaken to evaluate the potential utility of basic fibroblast growth factor in the induction of angiogenesis and osseous healing in bone previously exposed to high doses of irradiation. Thirty New Zealand rabbits were evaluated by introducing basic fibroblast growth factor into irradiated mandibular resection sites either prior to or simultaneous with reconstruction by corticocancellous autografts harvested from the ilium. The fate of the free bone grafts was then evaluated at 90 days postoperatively by microangiographic, histologic, and fluorochrome bone-labeling techniques. Sequestration, necrosis, and failure to heal to recipient osseous margins was observed both clinically and histologically in all nontreated irradiated graft sites as well as those receiving simultaneous angiogenic stimulation at the time of graft placement. No fluorescent activity was seen in these graft groups. In the recipient sites pretreated with basic fibroblast growth factor prior to placement of the graft, healing and reestablishment of mandibular contour occurred in nearly 50 percent of the animals. Active bone formation was evident at cortical margins adjacent to the recipient sites but was absent in the more central cancellous regions of the grafts.

  7. Effect of Surface Chemical Functionalities on Collagen Deposition by Primary Human Dermal Fibroblasts.

    PubMed

    Bachhuka, Akash; Hayball, John; Smith, Louise E; Vasilev, K

    2015-10-28

    Surface modification has been identified as an important technique that could improve the response of the body to implanted medical devices. Collagen production by fibroblasts is known to play a vital role in wound healing and device fibrous encapsulation. However, how surface chemistry affects collagen I and III deposition by these cells has not been systematically studied. Here, we report how surface chemistry influences the deposition of collagen I and III by primary human dermal fibroblasts. Amine (NH3), carboxyl acid (COOH), and hydrocarbon (CH3) surfaces were generated by plasma deposition. This is a practically relevant tool to deposit a functional coating on any type of substrate material. We show that fibroblasts adhere better and proliferate faster on amine-rich surfaces. In addition, the initial collagen I and III production is greater on this type of coating. These data indicates that surface modification can be a promising route for modulating the rate and level of fibrous encapsulation and may be useful in informing the design of implantable biomedical devices to produce more predictable clinical outcomes. PMID:26457649

  8. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    NASA Astrophysics Data System (ADS)

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili

    2016-04-01

    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  9. The effects of maternal iron deficiency on infant fibroblast growth factor-23 and mineral metabolism.

    PubMed

    Braithwaite, V S; Prentice, A; Darboe, M K; Prentice, A M; Moore, S E

    2016-02-01

    Fibroblast growth factor-23 (FGF23), a phosphate(Phos)-regulating hormone, is abnormally elevated in hypophosphataemic syndromes and an elevated FGF23 is a predictor of mortality in kidney disease. Recent findings suggest iron deficiency as a potential mediator of FGF23 expression and murine studies have shown in utero effects of maternal iron deficiency on offspring FGF23 and phosphate metabolism. Our aim was to investigate the impact of maternal iron status on infant FGF23 and mineral metabolites over the first 2years of life. Infants born to mothers with normal (NIn=25,) and low (LIn=25) iron status during pregnancy, from a mother-infant trial (ISRCTN49285450) in rural Gambia, West Africa, had blood and plasma samples analysed at 12, 24, 52, 78 and 104weeks (wk) of age. Circulating intact-FGF23 (I-FGF23), Phos, total alkaline phosphatase (TALP) and haemoglobin (Hb) decreased and estimated glomerular filtration rate increased over time [all P≤0.0001)]. C-terminal-FGF23 (C-FGF23) and TALP were significantly higher in LI compared with NI, from 52wk for C-FGF23 [Beta coefficient (SE) 18.1 (0.04) %, P=0.04] and from 24wk for TALP [44.7 (29.6) U/L, P=0.04]. Infant Hb was the strongest negative predictor of C-FGF23 concentration [-21% (4%) RU/mL, P≤0.0001], Phos was the strongest positive predictor of I-FGF23 [32.0(3.9) pg/mL, P≤0.0001] and I-FGF23 did not predict C-FGF23 over time [-0.5% (0.5%), P=0.3]. In conclusion, this study suggests that poor maternal iron status is associated with a higher infant C-FGF23 and TALP but similar I-FGF23 concentrations in infants and young children. These findings further highlight the likely public health importance of preventing iron deficiency during pregnancy. Whether or not children who are born to iron deficient mothers have persistently high concentrations of these metabolites and are more likely to be at risk of impaired bone development and pre-disposed to rickets requires further research. PMID:26453792

  10. Effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts in vitro

    PubMed Central

    ZHANG, HAI-YUAN; LIU, RUI; XING, YONG-JUN; XU, PING; LI, YAN; LI, CHEN-JUN

    2013-01-01

    This study aimed to investigate the effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts (HPLFs) at various times in vitro in order to further study plateau-hypoxia-induced periodontal disease. HPLFs (fifth passage) cultured by the tissue culture method were assigned to the slight (5% O2), middle (2% O2), and severe hypoxia (1% O2) groups and the control (21% O2) group, respectively. At 12, 24, 48 and 72 h, the proliferation and alkaline phosphatase (ALP) activities were detected. The ultrastructure of the severe hypoxia group was observed. HPLFs grew more rapidly with an increase in the degree of hypoxia at 12 and 24 h, and significant levels of proliferation (P<0.05) were observed in the severe hypoxia group at 24 h. Cell growth was restrained with an increase in the degree of hypoxia at 48 and 72 h, and the restrictions were clear (P<0.05) in the middle and severe hypoxia groups. ALP activity was restrained with increasing hypoxia at each time point. The restrictions were marked (P<0.05) in the severe hypoxia group at 24 h and in the middle and severe hypoxia groups at 48 and 72 h. However, the restriction was more marked (P<0.05) in the severe hypoxia group at 72 h. An increase was observed in the number of mitochondria and rough endoplasmic reticula (RER), with slightly expanded but complete membrane structures, in the severe hypoxia group at 24 h. At 48 h, the number of mitochondria and RER decreased as the mitochondria increased in size. Furthermore, mitochondrial cristae appeared to be vague, and a RER structural disorder was observed. At 72 h, the number of mitochondria and RER decreased further when the mitochondrial cristae were broken, vacuolar degeneration occurred, and the RER particles were reduced while the number of lysosomes increased. HPLF proliferation and mineralization was restrained. Additionally, HPLF structure was broken for a relatively long period of time in the middle and

  11. Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro

    PubMed Central

    Wermuth, Peter J.; Addya, Sankar; Jimenez, Sergio A.

    2011-01-01

    Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel

  12. Glucose and carbachol activate phospholipase C in digitonin-permeabilized islets

    SciTech Connect

    Wolf, B.A.; Florholmen, J.; Turk, J.; McDaniel, M.L.

    1987-05-01

    Stimulation of intact islets with D-glucose, the major insulin secretagogue, or with carbachol, a muscarinic agonist, results in the accumulation of inositoltrisphosphate (IP/sub 3/) suggesting that activation of phospholipase C (PLC) has a major role in stimulus-secretion coupling. Carbachol activation of PLC is an example of receptor-mediated activation in islets, whereas, the mechanism of glucose activation of PLC is controversial since a glucose receptor has not been identified. They have measured PLC activity in digitonin-permeabilized islets. Islets were labeled with /sup 3/H-inositol, permeabilized and IP/sub 3/ accumulation measured by HPLC. Carbachol, in the presence of ATP, GTP and 1 ..mu..M free Ca/sup 2 +/ released two-fold more Ins 1,3,4-P/sub 3/ than control in a time-dependent manner. Glucose, under the same conditions also significantly released more Ins 1,3,4-P/sub 3/ than control. This effect was not due to metabolism of glucose nor to an effect on the IP/sub 3/-phosphomonoesterase. Preliminary Ca/sup 2 +/-dependency studies indicate that PLC is not activated by Ca/sup 2 +/ in the submicromolar range. In conclusion, these studies show that Ca/sup 2 +/ does not activate PLC, and furthermore, that D-glucose may be recognized directly by PLC.

  13. Dickkopf-1 has an Inhibitory Effect on Mesenchymal Stem Cells to Fibroblast Differentiation

    PubMed Central

    Li, Yan; Qiu, Sang-Sang; Shao, Yan; Song, Hong-Huan; Li, Gu-Li; Lu, Wei; Zhu, Li-Mei

    2016-01-01

    Background: Mesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs (DKKs) are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6). DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway. Methods: Stable MSCs were randomly divided into four groups: MSCs control, MSCs + transforming growth factor-β (TGF-β), MSCs + DKK1, and MSCs + TGF-β + DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test. Immunofluorescence was used to detect protein expression in the Wnt/β-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression of fibroblast surface markers and Wnt/β-catenin signaling proteins. Results: Cultivated MSCs were found to conform to the characteristics of standard MSCs: expression of cluster of differentiation (CD) 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs + TGF-β. However, the expression was lower in the MSCs + TGF-β + DKK1. Immunofluorescence showed high expression of all Wnt/β-catnin molecules in the MSCs + TGF-β group but expressed in lower quantities in MSCs + TGF-β + DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/β-catenin signaling proteins β-catenin, T-cell factor

  14. Pharmacological and toxicological effects of co-exposure of human gingival fibroblasts to silver nanoparticles and sodium fluoride

    PubMed Central

    Inkielewicz-Stepniak, Iwona; Santos-Martinez, Maria Jose; Medina, Carlos; Radomski, Marek W

    2014-01-01

    Background Silver nanoparticles (AgNPs) and fluoride (F) are pharmacological agents widely used in oral medicine and dental practice due to their anti-microbial/anti-cavity properties. However, risks associated with the co-exposure of local cells and tissues to these xenobiotics are not clear. Therefore, we have evaluated the effects of AgNPs and F co-exposure on human gingival fibroblast cells. Methods Human gingival fibroblast cells (CRL-2014) were exposed to AgNPs and/or F at different concentrations for up to 24 hours. Cellular uptake of AgNPs was examined by transmission electron microscopy. Downstream inflammatory effects and oxidative stress were measured by real-time quantitative polymerase chain reaction (PCR) and reactive oxygen species (ROS) generation. Cytotoxicity and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and real-time quantitative PCR and flow cytometry, respectively. Finally, the involvement of mitogen-activated protein kinases (MAPK) was studied using Western blot. Results We found that AgNPs penetrated the cell membrane and localized inside the mitochondria. Co-incubation experiments resulted in increased oxidative stress, inflammation, and apoptosis. In addition, we found that co-exposure to both xenobiotics phosphorylated MAPK, particularly p42/44 MAPK. Conclusion A combined exposure of human fibroblasts to AgNPs and F results in increased cellular damage. Further studies are needed in order to evaluate pharmacological and potentially toxicological effects of AgNPs and F on oral health. PMID:24729703

  15. Insulin alters cAMP-activated lipolysis but not cAMP-inhibited glycogen synthase in permeabilized adipocytes

    SciTech Connect

    Mooney, R.A.; Wisniewski, J.L.

    1986-05-01

    Lipolysis and, to a lesser extent, glycogen synthase activity are regulated in adipocytes by cellular cAMP and counter-regulated by insulin. These activities were measured in situ in digitonin (20 ..mu..g/ml) permeabilized rat adipocytes. Incorporation of /sup 3/H UDP-glucose into endogenous glycogen in the presence of KF, EDTA and 10mM glucose-6-phosphate was the basis of the G.S. assay. Cellular GS activity determined by this technique was 1.4 +/- 0.2 fold greater than that of matched homogenates. Insulin treatment of intact cells prior to permeabilization increased GS activity ratio (-/+ G-6-P) 2.5 fold when subsequently measured by the in situ assay. Following digitonin permeabilization, addition of cAMP to the suspension medium increased lipolysis 7 fold and decreased GS activity ratio to 0.38 +/- 0.01 from a basal value of 0.44 +/- 0.06. ATP had a negligible effect on lipolysis but decreased GS to 0.16 +/- 0.04. ATP plus cAMP was only slightly more effective on GS than ATP alone. Insulin at 10/sup -9/M inhibited cAMP-dependent lipolysis by 27% but had no effect on the cAMP- or ATP-dependent decrease in GS. These results suggest that insulin's counter-regulatory mechanisms on these two cAMP-dependent processes may be different.

  16. Proliferative effect of green light emitting diode irradiation on chicken fibroblasts in hyperglycaemic circumstances: a preliminary in vitro study

    NASA Astrophysics Data System (ADS)

    Vinck, Elke; Cagnie, Barbara; Declercq, Heidi; Cornelissen, Ria; Cambier, Dirk

    2004-09-01

    A reduced mortality due to hyperglycaemia was noted since the development of insulin treatment for type I diabetes and various oral hypoglycaemic agents for type II diabetes. Nevertheless the chronic metabolic disorder, Diabetes Mellitus, remains an important cause of morbidity and mortality due to a series of common secondary metabolic complications. Patients with diabetes have an increased tendency to develop infections of the skin. Healing of skin lesions in diabetics evolves often relatively slow and the lesions tend to be more severe than in non-diabetics. Endeavouring to accelerate the healing process of skin lesions in diabetic patients, this preliminary in vitro study investigates the efficacy of green Light Emitting Diode (LED) irradiation on fibroblast proliferation of cells in hyperglycaemic circumstances. In an attempt to imitate the diabetic environment, embryonic chicken fibroblasts were cultured in hyperglycaemic medium (30.000mg Glucose per litre Hanks Medium). LED irradiation was performed three consecutive days with a wavelength of 540 nm and a power output of 10 mW, at 0,6 cm distance from the fibroblasts. Each treatment lasted 3 minutes, resulting in a surface energy density of 0,2 J/cm2. Statistical analysis revealed that LED irradiation at the applied parameters induced a higher rate of proliferation in hyperglycaemic circumstances after irradiation than in the same circumstances without irradiation. Regarding these results the effectiveness of green LED irradiation on cells in hyperglycaemic circumstances is proven. To ensure the effectiveness and to evaluate the value of LED irradiation in vivo, further research is required.

  17. Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes

    PubMed Central

    Heidi Au, Hoi Ting; Cheng, Irene; Chowdhury, Mohammad Fahad; Radisic, Milica

    2007-01-01

    In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1 to 80μm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13μm, and the average depth in the range from 140nm to 700nm (AFM). The surfaces with abrasions 13μm wide and 700nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1ms duration, 1Hz, 2.3V/cm or 4.6V/cm) was initiated 24hr after cell seeding and maintained for additional 72hr. The cover slips were positioned between the carbon rod electrodes so that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability (live/dead staining). The presence of a well developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical

  18. Growth Inhibition and Membrane Permeabilization of Candida lusitaniae Using Varied Pulse Shape Electroporation

    PubMed Central

    Novickij, V.; Grainys, A.; Lastauskienė, E.; Kananavičiūtė, R.; Pamedytytė, D.; Zinkevičienė, A.; Kalėdienė, L.; Novickij, J.; Paškevičius, A.; Švedienė, J.

    2015-01-01

    Candida lusitaniae is an opportunistic yeast pathogen, which can readily develop resistance to antifungal compounds and result in a complex long-term treatment. The efficient treatment is difficult since structure and metabolic properties of the fungal cells are similar to those of eukaryotic host. One of the potential methods to improve the inhibition rate or the cell permeability to inhibitors is the application of electroporation. In this work we investigated the dynamics of the growth inhibition and membrane permeabilization of C. lusitaniae by utilizing the various pulse shape and duration electric field pulses. Our results indicated that single electroporation procedure using 8 kV/cm electric field may result in up to 51 ± 5% inhibition rate. Also it has been experimentally shown that the electroporation pulse shape may influence the inhibitory effect; however, the amplitude of the electric field and the pulse energy remain the most important parameters for definition of the treatment outcome. The dynamics of the cell membrane permeabilization in the 2–8 kV/cm electric field were overviewed. PMID:26697485

  19. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation

    PubMed Central

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-01-01

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05) in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome. PMID:27120618

  20. Tight Coupling of Na+/K+-ATPase with Glycolysis Demonstrated in Permeabilized Rat Cardiomyocytes

    PubMed Central

    Sepp, Mervi; Sokolova, Niina; Jugai, Svetlana; Mandel, Merle; Peterson, Pearu; Vendelin, Marko

    2014-01-01

    The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis - sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose. PMID:24932585

  1. Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells.

    PubMed

    Anmann, Tiia; Eimre, Margus; Kuznetsov, Andrey V; Andrienko, Tatiana; Kaambre, Tuuli; Sikk, Peeter; Seppet, Evelin; Tiivel, Toomas; Vendelin, Marko; Seppet, Enn; Saks, Valdur A

    2005-06-01

    The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system. PMID:15955072

  2. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    PubMed

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-01-01

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05) in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome. PMID:27120618

  3. Effects of donor fibroblasts expressing OCT4 on bovine embryos generated by somatic cell nuclear transfer.

    PubMed

    Goissis, Marcelo D; Suhr, Steven T; Cibelli, Jose B

    2013-02-01

    The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos. PMID:23276226

  4. Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

    PubMed Central

    Heidari, Mahmoud; Malekshah, Abbasali Karimpour; Parivar, Kazem; Khanbabaei, Ramezan; Rafiei, Alireza

    2011-01-01

    Background The aim of this study was to evaluate fibroblast co-culture on in vitro maturation and fertilization of prepubertal mouse preantral follicles. Materials and Methods The ovaries of 12-14 day old mice were dissected and 120-150 μm intact preantral follicles with one or two layers of granulosa cells, and round oocytes were cultured individually in α-minimal essential medium (α-MEM) supplemented with 5% fetal bovine serum (FBS), 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin, selenium mix, 100 μg/ml penicillin and 50 μg/ml streptomycin as base medium for 12 days. A total number of 226 follicules were cultured under two conditions: i) base medium as control group (n=113); ii) base medium co-cultured with mouse embryonic fibroblast (MEF) (n=113). Follicular diameters, alone, in addition to other factors were analyzed by student’s t-test and chi-square test, respectively. Results The co-culture group showed significant differences (p<0.05) in growth rate (days 4, 6 and 8 of the culture period) and survival rate. However, there was no significant difference in antrum formation, ovulation rate and embryonic development of released oocytes. There were significant differences (p<0.05) in the estradiol and progesterone secretion at all days between the co-culture and control groups. Conclusion Fibroblast co-culture increased survival rate and steroid production of preantral follicles by promoting granulosa cell proliferation. PMID:24917917

  5. Effects of the Nd:YAG laser and combined treatments on in vitro fibroblast attachment to root surfaces.

    PubMed

    Thomas, D; Rapley, J; Cobb, C; Spencer, P; Killoy, W

    1994-01-01

    The purpose of this in vitro study was to evaluate the effects of the Nd:YAG laser either alone or in combination with root planning or air-powder abrasive treatment on fibroblast attachment to non-diseased root surfaces. 28, 4 x 4 mm root specimens and four disc-shaped root specimens 6 mm in diameter were obtained from unerupted 3rd molars. The root segments were randomly assigned to 4 treatment groups: (1) control; (2) laser-only treated; (3) laser treated followed by root planning; (4) laser treated followed by air-powder abrasive treatment. Laser-treated root specimens were exposed for 1 min with the Nd:YAG laser calibrated at an energy setting of 75 mJ at 20 pulses/s using a 320 microns contact fiber. The contact fiber was held parallel to the root segments and the root segments were kept moist with distilled water. Following the prescribed treatments, the root specimens were incubated with fibroblast cultures and then prepared for SEM examination. Results of cell counts of fibroblasts attached to specimens within each treatment group yielded the following means and standard deviations: control groups, 181.64 +/- 44.74; lased only, 78.57 +/- 21.35; lased and root planed 125.35 +/- 26.13; and lased followed by an air-powder abrasive, 177.28 +/- 55.71. Application of ANOVA followed by the Dunn Multiple Comparison test revealed significant differences (p < 0.01) in the number of attached cells between the control and laser-only treated groups; and between the laser-only and laser/air-powder abrasive treated groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8126242

  6. Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

    SciTech Connect

    Słonina, Dorota; Biesaga, Beata; Janecka, Anna; Kabat, Damian; Bukowska-Strakova, Karolina; Gasińska, Anna

    2014-02-01

    Purpose: In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. Methods and Materials: The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. Results: The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. Conclusions: The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis.

  7. mir-1-mediated paracrine effect of cancer-associated fibroblasts on lung cancer cell proliferation and chemoresistance.

    PubMed

    Li, Jianmin; Guan, Jing; Long, Xiaoping; Wang, Yang; Xiang, Xudong

    2016-06-01

    Lung cancer is the leading cause of cancer-related mortality in humans worldwide. Moreover, the overall 5-year survival rate is only 15%. Pathologically almost 80% of all lung cancer cases are non-small cell lung cancer (NSCLC). Cancer-associated fibroblasts (CAFs) have been found to exist in a large number of NSCLCs. CAFs have been proven to promote tumor progression, metastasis and resistance to therapy through paracrine effects in most solid tumors. In the present study, firstly we isolated CAFs from patient tissues and demonstrated that they promoted cell proliferation and chemoresistance to cisplatin in the lung cancer cell lines A549 and 95D in a paracrine manner. Secondly, using ELISA and quantative PCR, we found that a higher amount of stromal cell-derived factor 1 (SDF-1) existed in the CAFs rather than that observed in the normal fibroblasts (NFs). Thirdly, we detected that SDF-1 facilitated lung cancer cell proliferation and drug resistance via the CXCR4-mediated signaling pathway which involved NF-κB and Bcl-xL. Moreover, we also confirmed that the expression level of SDF-1 in the CAFs was negatively regulated by microRNA mir-1 through microRNA overexpression and quantitative PCR. Overall, our data provide one explanation for the effects of CAFs on lung cancer cells. Meanwhile, our results also suggest CAFs as a potential therapeutic target in tumor treatment. PMID:27035564

  8. Effects of Abnormal Savda Munzip on the Proliferation Activity and Migration Ability of Fibroblasts Derived from Hypertrophic Scar In Vitro

    PubMed Central

    Wang, Hujun; Gao, Weicheng; Kong, Menglong; Li, Nan; Shaolin, Ma

    2015-01-01

    Background. To explore the effect of ASMq on proliferation and migration ability of the fibroblast derived from HS of donor (HSFbs) in vitro. Methods. The HSFbs were cultured from tissue specimens and passaged to the 3~4 generation, which were treated with the different concentrations of ASMq and 5-Fu from 1 to 11 days. The difference of HSFbs proliferation activity was analyzed by the CCK-8 method. The HSFbs migration ability in ASMq (0.4 mg/mL) was analyzed by the Cell Scratch method. Results. Transmission electron microscope result shows ASMq concentration significantly increases and fibroblast cell structure markedly change in the experimental group. The proliferation activity of the HSFbs was obviously weakened in ASMq groups than those of the group A (P < 0.05) at seven days. The group C (0.4 mg/mL) is better suitable than other three ASMq treatment groups. Cell Migration Assay shows that the migration ability HSFbs was significantly reduced in ASMq (0.4 mg/mL) treatment group compared with those of blank control group at both 24 h and 48 h (P < 0.05). Conclusions. These results suggest that ASMq effectively restrains the proliferation and migration ability of the HTSFbs in vitro, which can be one of the mechanisms for the prevention and treatment of HS. PMID:25821502

  9. Effect of CTRP3 on activation of adventitial fibroblasts induced by TGF-β1 from rat aorta in vitro

    PubMed Central

    Lin, Shaohui; Ma, Shaojun; Lu, Ping; Cai, Wenwei; Chen, Yi; Sheng, Jing

    2014-01-01

    CTRP3, discovered as novel adipokines, is a member of the C1q tumor necrosis factor (TNF) related protein (CTRP) super-family. CTRP3 is found to function as adipokines that display diverse biological activities in metabolic and cardiovascular diseases. Recent study demonstrated that CTRP3 was protective against pathological cardiac remodeling in mice. Nevertheless, the effect of CTRP3 on vascular remodeling remains undefined. Our present study aimed to explore the effects of adipokine CTRP3 on the activation of adventitial fibroblasts (AFs) induced by TGF-β1. Immunofluorescent staining, real-time PCR and Western blot were conducted to evaluate the expression of α-smooth muscle-actin (α-SMA) and collagen I. The expression of CTGF was evaluated by enzymelinked immunosorbent assay (ELISA), while the proliferation and migration of adventitial fibroblasts were detected by using cell counting kit-8 (CCK-8) assay and Transwell technique, respectively. Functional analysis showed that CTRP3 inhibited TGF-β1 inducing AFs phenotypic conversion, collagen synthesis, proliferation and migration. The secretion of CTGF was also inhibited by CTRP3. Our findings suggest that CTRP3 may be beneficial to the prevention of cardiovascular diseases and provide a promising therapeutic strategy to attenuate vascular remodeling. PMID:24966928

  10. Cytoprotective effects of cerium and selenium nanoparticles on heat-shocked human dermal fibroblasts: an in vitro evaluation

    PubMed Central

    Yuan, Bo; Webster, Thomas J; Roy, Amit K

    2016-01-01

    It is a widely accepted fact that environmental factors affect cells by modulating the components of subcellular compartments and altering metabolic enzymes. Factors (such as oxidative stress and heat-shock-induced proteins and heat shock factors, which upregulate stress-response related genes to protect affected cells) are commonly altered during changes in environmental conditions. Studies by our group and others have shown that nanoparticles (NPs) are able to efficiently attenuate oxidative stress by penetrating into specific tissues or organs. Such findings warrant further investigation on the effects of NPs on heat-shock-induced stress, specifically in cells in the presence or absence (pretreated) of NPs. Here, we examined the cytoprotective effects of two different NPs (cerium and selenium) on heat-induced cell death for a model cell using dermal fibroblasts. We report for the first time that both ceria and selenium NPs (at 500 µg/mL) possess stress-relieving behavior on fibroblasts undergoing heat shock. Such results indicate the need to further develop these NPs as a novel treatment for heat shock. PMID:27103800

  11. Effect of structure, topography and chemistry on fibroblast adhesion and morphology.

    PubMed

    Mateos-Timoneda, Miguel A; Castano, Oscar; Planell, Josep A; Engel, Elisabeth

    2014-07-01

    Surface biofunctionalisation of many biodegradable polymers is one of the used strategies to improve the biological activity of such materials. In this work, the introduction of collagen type I over the surface of a biodegradable polymer (poly lactic acid) processed in the forms of films and fibers leads to an enhancing of the cellular adhesion of human dermal fibroblast when compared to unmodified polymer and biomolecule-physisorbed polymer surface. The change of topography of the material does not affect the cellular adhesion but results in a higher proliferation of the fibroblast cultured over the fibers. Moreover, the difference of topography governs the cellular morphology, i.e. cells adopt a more stretched conformation where cultured over the films while a more elongated with lower area morphology are obtained for the cells grown over the fibers. This study is relevant for designing and modifying different biodegradable polymers for their use as scaffolds for different applications in the field of Tissue Engineering and Regenerative Medicine. PMID:24668270

  12. Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

    PubMed

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Kim, Jin Wook; Lee, Tae Hee; Lee, Byeong Chun

    2015-09-01

    Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P < 0.05). In vivo developmental potential of cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P < 0.05). The culture medium can induce changes in gene expression of donor cells and telomerase activity, and these alterations can also affect in vivo developmental competence of the cloned embryos. PMID:26001598

  13. Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

    PubMed

    Moruzzi, Noah; Del Sole, Marianna; Fato, Romana; Gerdes, Jantje M; Berggren, Per-Olof; Bergamini, Christian; Brismar, Kerstin

    2014-08-01

    High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia. PMID:24814290

  14. Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed. PMID:27140915

  15. Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

    PubMed Central

    Bunthof, Christine J.; van Schalkwijk, Saskia; Meijer, Wilco; Abee, Tjakko; Hugenholtz, Jeroen

    2001-01-01

    A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed. PMID:11526032

  16. Fluorescent method for monitoring cheese starter permeabilization and lysis.

    PubMed

    Bunthof, C J; van Schalkwijk, S; Meijer, W; Abee, T; Hugenholtz, J

    2001-09-01

    A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed. PMID:11526032

  17. Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.

    PubMed

    Ruszová, Ema; Cheel, José; Pávek, Stanislav; Moravcová, Martina; Hermannová, Martina; Matějková, Ilona; Spilková, Jiřina; Velebný, Vladimír; Kubala, Lukáš

    2013-09-01

    Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits. PMID:23817638

  18. Anti-wrinkle effects of a tuna heart H2O fraction on Hs27 human fibroblasts

    PubMed Central

    KIM, YOUNG-MIN; JUNG, HEE-JIN; CHOI, JAE-SUE; NAM, TAEK-JEONG

    2016-01-01

    With the increase in life expectancy, there is also growing interest in anti-aging treatments and technologies. The development of anti-aging functional drugs for the skin, and foods from natural sources, may offer solutions to this global matter. Aging involves structural, functional and biochemical changes that occur throughout cells and bodily tissues; the amount of hormones secreted from of all human organs, including the skin, decreases over time. Matrix metalloproteinase (MMP) genes (MMP-1 and -8) play an important role in the aging of skin fibroblasts. For example, an increased MMP expression causes accelerated aging and the degradation of skin elasticity-related genes. In the present study, we examined the anti-wrinkle effects of tuna heart extract which are mediated through the inhibition of MMPs in skin cells. Generally, tuna contains high concentrations of selenium and antioxidants, which serve to remove free radicals, and is known to delay skin and body aging. In addition, unsaturated fatty acids in tuna help to maintain the natural glossy look of skin, and increase skin elasticity, providing moisture for dry skin. A recent study confirmed the various bio-effects of boiled tuna extract and muscle. However, bioactivity studies using tuna heart are limited. Thus, in the present study, we obtained extracts and fractions of tuna heart, and examined their effects on Hs27 human fibroblast proliferation using an MTS assay. In addition, we measured procollagen type 1 levels and elastase activity, and performed β-galactosidase staining. We then measured the expression levels of phosphatidylinositol 3-kinase/Akt and MMP-related genes by western blot analysis and RT-PCR. Our results revealed that tuna heart extract decreased MMP expression by upregulating tissue inhibitors of metallopro-teinase-1 (TIMP-1) and decreasing elastase activity, thus exerting anti-aging and anti-wrinkle effects by increasing collagen synthesis and promoting skin fibroblast proliferation

  19. Anti-wrinkle effects of a tuna heart H2O fraction on Hs27 human fibroblasts.

    PubMed

    Kim, Young-Min; Jung, Hee-Jin; Choi, Jae-Sue; Nam, Taek-Jeong

    2016-01-01

    With the increase in life expectancy, there is also growing interest in anti-aging treatments and technologies. The development of anti-aging functional drugs for the skin, and foods from natural sources, may offer solutions to this global matter. Aging involves structural, functional and biochemical changes that occur throughout cells and bodily tissues; the amount of hormones secreted from of all human organs, including the skin, decreases over time. Matrix metalloproteinase (MMP) genes (MMP-1 and -8) play an important role in the aging of skin fibroblasts. For example, an increased MMP expression causes accelerated aging and the degradation of skin elasticity-related genes. In the present study, we examined the anti-wrinkle effects of tuna heart extract which are mediated through the inhibition of MMPs in skin cells. Generally, tuna contains high concentrations of selenium and antioxidants, which serve to remove free radicals, and is known to delay skin and body aging. In addition, unsaturated fatty acids in tuna help to maintain the natural glossy look of skin, and increase skin elasticity, providing moisture for dry skin. A recent study confirmed the various bio-effects of boiled tuna extract and muscle. However, bioactivity studies using tuna heart are limited. Thus, in the present study, we obtained extracts and fractions of tuna heart, and examined their effects on Hs27 human fibroblast proliferation using an MTS assay. In addition, we measured procollagen type 1 levels and elastase activity, and performed β-galactosidase staining. We then measured the expression levels of phosphatidylinositol 3-kinase/Akt and MMP-related genes by western blot analysis and RT-PCR. Our results revealed that tuna heart extract decreased MMP expression by upregulating tissue inhibitors of metalloproteinase-1 (TIMP-1) and decreasing elastase activity, thus exerting anti-aging and anti-wrinkle effects by increasing collagen synthesis and promoting skin fibroblast

  20. Effects of lunar and mars dust on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Brix, Klaudia; Slenzka, Klaus; Rehders, Maren; Sadhukhan, Annapurna; Mistry, Rima; Duenne, Matthias; Kempf, Juergen

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respira-tory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of lunar dust on human health is required to best support future missions to moon. In this study, we used different methods to assess the specific effects of lunar dust onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and since a well orchestrated program ensures proper repair in cases of wounding. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology, metabolic state, survival and proliferation of the cells were determined. Cytotoxi-city and proliferation were measured using the MTT assay, metabolic activity was analyzed by vital staining of mitochondria, and phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells. It was found that the effects of the two types of soils on the different features of both cell lines varied to considerable extent, and that lunar and mars dust were specific in their effects. The obtained results will facilitate detailed inves-tigations of dust exposure during wound healing and will ease risk assessment studies for e.g. lunar lander approaches. The investigations will help to assess the risks and to determine safety measures to be taken during extraterrestrial expeditions in order to minimize risks to human health associated with exposure of human skin to dust contaminants.

  1. The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts.

    PubMed

    Martin, C E; Croft, K D; van Bockxmeer, F M; Constable, I J

    1987-12-10

    Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye. PMID:3118960

  2. Effects of Cellular Pathway Disturbances on Misfolded Superoxide Dismutase-1 in Fibroblasts Derived from ALS Patients

    PubMed Central

    Keskin, Isil; Forsgren, Elin; Lange, Dale J.; Weber, Markus; Birve, Anna; Synofzik, Matthis; Gilthorpe, Jonathan D.; Andersen, Peter M.; Marklund, Stefan L.

    2016-01-01

    Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS. PMID:26919046

  3. Extract of Ettlia sp. YC001 Exerts Photoprotective Effects against UVB Irradiation in Normal Human Dermal Fibroblasts.

    PubMed

    Lee, Jeong-Ju; An, Sungkwan; Kim, Ki Bbeum; Heo, Jina; Cho, Dae-Hyun; Oh, Hee-Mock; Kim, Hee-Sik; Bae, Seunghee

    2016-04-28

    The identification of novel reagents that exert a biological ultraviolet (UV)-protective effect in skin cells represents an important strategy for preventing UV-induced skin aging. To this end, we investigated the potential protective effects of Ettlia sp. YC001 extracts against UV-induced cellular damage in normal human dermal fibroblasts (NHDFs). We generated four different extracts from Ettlia sp. YC001, and found that they exhibit low cytotoxicity in NHDFs. The ethyl acetate extract of Ettlia sp. YC001 markedly decreased UVB-induced cytotoxicity. Additionally, the ethyl acetate extract significantly inhibited the production of hydrogen peroxide-induced reactive oxygen species. Moreover, it inhibited UVB-induced thymine dimers, as confirmed by luciferase assay and thymine dimer dot-blot assay. Thus, the study findings suggest Ettlia sp. YC001 extract as a novel photoprotective reagent on UVB-induced cell dysfunctions in NHDFs. PMID:26718469

  4. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  5. Anti-biofilm activity of chitosan gels formulated with silver nanoparticles and their cytotoxic effect on human fibroblasts.

    PubMed

    Pérez-Díaz, M; Alvarado-Gomez, E; Magaña-Aquino, M; Sánchez-Sánchez, R; Velasquillo, C; Gonzalez, C; Ganem-Rondero, A; Martínez-Castañon, G; Zavala-Alonso, N; Martinez-Gutierrez, F

    2016-03-01

    The development of multi-species biofilms in chronic wounds is a serious health problem that primarily generates strong resistance mechanisms to antimicrobial therapy. The use of silver nanoparticles (AgNPs) as a broad-spectrum antimicrobial agent has been studied previously. However, their cytotoxic effects limit its use within the medical area. The purpose of this study was to evaluate the anti-biofilm capacity of chitosan gel formulations loaded with AgNPs, using silver sulfadiazine (SSD) as a standard treatment, on strains of clinical isolates, as well as their cytotoxic effect on human primary fibroblasts. Multi-species biofilm of Staphylococcus aureus oxacillin resistant (MRSA) and Pseudomonas aeruginosa obtained from a patient with chronic wound infection were carried out using a standard Drip Flow Reactor (DFR) under conditions that mimic the flow of nutrients in the human skin. Anti-biofilm activity of chitosan gels and SSD showed a log-reduction of 6.0 for MRSA when chitosan gel with AgNPs at a concentration of 100 ppm was used, however it was necessary to increase the concentration of the chitosan gel with AgNPs to 1000 ppm to get a log-reduction of 3.3, while the SSD showed a total reduction of both bacteria in comparison with the negative control. The biocompatibility evaluation on primary fibroblasts showed better results when the chitosan gels with AgNPs were tested even in the high concentration, in contrast with SSD, which killed all the primary fibroblasts. In conclusion, chitosan gel formulations loaded with AgNPs effectively prevent the formation of biofilm and kill bacteria in established biofilm, which suggest that chitosan gels with AgNPs could be used for prevention and treatment of infections in chronic wounds. The statistic significance of the biocompatibility of chitosan gel formulations loaded with AgNPs represents an advance; however further research and development are necessary to translate this technology into therapeutic and

  6. Photobiomodulation of distinct lineages of human dermal fibroblasts: a rational approach towards the selection of effective light parameters for skin rejuvenation and wound healing

    NASA Astrophysics Data System (ADS)

    Mignon, Charles; Uzunbajakava, Natallia E.; Raafs, Bianca; Moolenaar, Mitchel; Botchkareva, Natalia V.; Tobin, Desmond J.

    2016-03-01

    Distinct lineages of human dermal fibroblasts play complementary roles in skin rejuvenation and wound healing, which makes them a target of phototherapy. However, knowledge about differential responses of specific cell lineages to different light parameters and moreover the actual molecular targets remain to be unravelled. The goal of this study was to investigate the impact of a range of parameters of light on the metabolic activity, collagen production, and cell migration of distinct lineages of human dermal fibroblasts. A rational approach was used to identify parameters with high therapeutic potential. Fibroblasts exhibited both inhibitory and cytotoxic change when exposed to a high dose of blue and cyan light in tissue culture medium containing photo-reactive species, but were stimulated by high dose red and near infrared light. Cytotoxic effects were eliminated by refreshing the medium after light exposure by removing potential ROS formed by extracellular photo-reactive species. Importantly, distinct lineages of fibroblasts demonstrated opposite responses to low dose blue light treatment when refreshing the medium after exposure. Low dose blue light treatment also significantly increased collagen production by papillary fibroblasts; high dose significantly retarded closure of the scratch wound without signs of cytotoxicity, and this is likely to have involved effects on both cell migration and proliferation. We recommend careful selection of fibroblast subpopulations and their culture conditions, a systematic approach in choosing and translating treatment parameters, and pursuit of fundamental research on identification of photoreceptors and triggered molecular pathways, while seeking effective parameters to address different stages of skin rejuvenation and wound healing.

  7. Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon's capsule compared with mitomycin C

    PubMed Central

    Heilmann, C.; Schonfeld, P.; Schluter, T.; Bohnensack, R.; Behrens-Baumann, W.

    1999-01-01

    BACKGROUND/AIMS—To investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon's capsule fibroblasts in comparison with the antitumour antibiotic mitomycin C.
METHODS—After determination of effective concentrations of idarubicin, fibroblasts of the human Tenon's capsule were exposed to idarubicin or mitomycin C at concentrations ranging from 0.1 µg/ml to 1 µg/ml or from 2.5 µg/ml to 250 µg/ml, respectively, for 0.5, 2, or 5 minutes and cultured for 60 days. Cell death by apoptosis caused by idarubicin treatment was confirmed by Hoechst 33258 staining. Further proliferation was explored by cell counting and by 3H-thymidine uptake. Protein synthesis was measured by 3H-proline uptake and motility was assessed by agarose droplet motility assay.
RESULTS—Idarubicin is able to exert toxicity and to induce apoptosis during a short time exposure of 0.5 minutes at concentrations of 0.3-1 µg/ml resulting in a significant reduction in cell number compared with the control after 60 days. For mitomycin C, higher concentrations and longer expositions were necessary. Even after treatment with 1 µg/ml idarubicin or 250 µg/ml mitomycin C a few cells were able to incorporate 3H-thymidine. 3H-proline uptake up to 10 days after exposure to 0.3 µg/ml idarubicin was found not to be decreased. Cell motility was reduced after treatment with 1 µg/ml idarubicin for 5 minutes or with 250 µg/ml mitomycin C for 2 or 5 minutes. For low mitomycin C concentrations, an increase in motility was found during the first 10 days.
CONCLUSION—Idarubicin reduces proliferation of human Tenons's capsule fibroblasts after incubation for 0.5 minutes at concentrations as low as 0.3-1 µg/ml. In comparison, mitomycin C requires longer exposure times and higher doses for equal results. Therefore, idarubicin may be useful in the prevention of glaucoma filtering surgery failure

  8. Mitochondrial permeabilization without caspase activation mediates the increase of basal apoptosis in cells lacking Nrf2.

    PubMed

    Ariza, Julia; González-Reyes, José A; Jódar, Laura; Díaz-Ruiz, Alberto; de Cabo, Rafael; Villalba, José Manuel

    2016-06-01

    Nuclear factor E2-related factor-2 (Nrf2) is a cap'n'collar/basic leucine zipper (b-ZIP) transcription factor which acts as sensor of oxidative and electrophilic stress. Low levels of Nrf2 predispose cells to chemical carcinogenesis but a dark side of Nrf2 function also exists because its unrestrained activation may allow the survival of potentially dangerous damaged cells. Since Nrf2 inhibition may be of therapeutic interest in cancer, and a decrease of Nrf2 activity may be related with degenerative changes associated with aging, it is important to investigate how the lack of Nrf2 function activates molecular mechanisms mediating cell death. Murine Embryonic Fibroblasts (MEFs) bearing a Nrf2 deletion (Nrf2KO) displayed diminished cellular growth rate and shortened lifespan compared with wild-type MEFs. Basal rates of DNA fragmentation and histone H2A.X phosphorylation were higher in Nrf2KO MEFs, although steady-state levels of reactive oxygen species were not significantly increased. Enhanced rates of apoptotic DNA fragmentation were confirmed in liver and lung tissues from Nrf2KO mice. Apoptosis in Nrf2KO MEFs was associated with a decrease of Bcl-2 but not Bax levels, and with the release of the mitochondrial pro-apoptotic factors cytochrome c and AIF. Procaspase-9 and Apaf-1 were also increased in Nrf2KO MEFs but caspase-3 was not activated. Inhibition of XIAP increased death in Nrf2KO but not in wild-type MEFs. Mitochondrial ultrastructure was also altered in Nrf2KO MEFs. Our results support that Nrf2 deletion produces mitochondrial dysfunction associated with mitochondrial permeabilization, increasing basal apoptosis through a caspase-independent and AIF-dependent pathway. PMID:27016073

  9. Procedure for the permeabilization and cryobiological preservation of Drosophila embryos

    SciTech Connect

    Cole, K.W.; Schreuders, P.D.; Mahowald, A.P.; Mazur, P. |

    1993-05-06

    The authors describe the detailed protocol developed in their laboratory at Oak Ridge for the permeabilization and cryobiological preservation of embryos of Drosophila melanogaster, Oregon R strain. The protocol is supplemented by notes containing two sorts of information. One category includes references to the appropriate portions of their published papers giving the scientific rationale and experimental basis for important steps. The other category is concerned with the criticality of certain steps and the precision with which they need to be performed. As an aid to investigators, the authors list even ordinary pieces of equipment. Brand names and model numbers are given where it is either important or convenient for readers to know precisely what is used.

  10. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis.

    PubMed

    Jourdain, Alexis; Martinou, Jean-Claude

    2009-10-01

    Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP. PMID:19439192

  11. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures.

    PubMed

    Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

    2016-01-01

    Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. PMID:27621603

  12. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    PubMed Central

    Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

    2016-01-01

    Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. PMID:27621603

  13. Inhibitory effects of PPARγ ligands on TGF-β1-induced CTGF expression in cat corneal fibroblasts.

    PubMed

    Jeon, Kye-Im; Phipps, Richard P; Sime, Patricia J; Huxlin, Krystel R

    2015-09-01

    Ligands of Peroxisome Proliferator Activated Receptor gamma (PPARγ) possess strong anti-fibrotic properties in the cornea and several other body tissues. In the cornea, we recently showed this class of molecules to prevent stromal myofibroblast differentiation partially by blocking the actions of p38 mitogen-activated protein kinase (MAPK). However, given the important role assigned to connective tissue growth factor (CTGF) in mediating corneal fibrosis, here we asked whether PPARγ ligands also act by affecting transforming growth factor-β (TGF-β) 1-induced expression of CTGF in cultured corneal fibroblasts. Corneal keratocytes were isolated from young, adult cats and early passage cells were exposed to TGF-β1 with or without the PPARγ ligands Rosiglitazone, Troglitazone and 15d-PGJ2. Western blots were used to assay levels of CTGF and alpha smooth muscle actin (αSMA), a marker of myofibroblast differentiation. CTGF siRNA demonstrated a critical role for CTGF in TGF-β1-mediated myofibroblast differentiation, while exogenously applied CTGF potentiated the pro-fibrogenic effects of TGF-β1. TGF-β1-mediated increases in CTGF and αSMA expression were strongly inhibited by all three PPARγ ligands tested, and by a c-jun N-terminal kinase (JNK) inhibitor. However, while extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (AKT) and p38 MAPK inhibitors also blocked TGF-β1-induced αSMA induction, they did not dampen TGF-β1-induced increases in levels of CTGF. Thus, we conclude that PPARγ ligands block TGF-β1-induced increases in CTGF levels in cat corneal fibroblasts. They appear to do this in addition to their anti-fibrotic effect on p38 MAPK, providing a second intracellular pathway by which PPARγ ligands block αSMA induction. PMID:26142957

  14. Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation.

    PubMed

    Rose, Laura C; Fitzsimmons, Ross; Lee, Poh; Krawetz, Roman; Rancourt, Derrick E; Uludağ, Hasan

    2013-05-01

    Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder-free monolayers, up to 40 ng/ml of exogenous bFGF did not support self-renewal of mES without LIF during cell expansion. During spontaneous differentiation in high-density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage-specific embryonic antigen-1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca-1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP-2. These results suggest that bFGF could be utilized to expand the cell population in high-density cultures in addition to enriching the BMP-2 responsiveness of mES cells. PMID:22674886

  15. The effect of amphiphilic siloxane oligomers on fibroblast and keratinocyte proliferation and apoptosis.

    PubMed

    Lynam, Emily C; Xie, Yan; Loli, Bree; Dargaville, Tim R; Leavesley, David I; George, Graeme A; Upton, Zee

    2010-11-01

    The formation of hypertrophic scars (HSF) is a frequent medical outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS) or apoptosis-inducing agents, including bleomycin. Although widely used, knowledge regarding SGS and their mode of action is limited. Preliminary research has shown that small amounts of amphiphilic silicone present in SGS have the ability to move into skin during treatment. We demonstrate herein that a commercially available analogue of these amphiphilic siloxane species, the rake copolymer GP226, decreases collagen synthesis on exposure to cultures of fibroblasts derived from HSF. By size exclusion chromatography, GP226 was found to be a mixture of siloxane species, containing five fractions of different molecular weight. By studies of collagen production, cell viability and proliferation, it was revealed that a low molecular weight fraction (fraction IV) was the most active, reducing the number of viable cells present after treatment and thereby reducing collagen production as a result. On exposure of fraction IV to human keratinocytes, viability and proliferation were also significantly affected. HSF undergoing apoptosis after application of fraction IV were also detected via real-time microscopy and by using the TUNEL assay. Taken together, these data suggests that these amphiphilic siloxanes could be potential non-invasive substitutes to apoptotic-inducing chemical agents that are currently used as scar treatments. PMID:20725963

  16. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed Central

    Higerd, T B; Vesole, D H; Goust, J M

    1978-01-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. Images PMID:689736

  17. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed

    Higerd, T B; Vesole, D H; Goust, J M

    1978-08-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. PMID:689736

  18. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application

    PubMed Central

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it. PMID:27597959

  19. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application.

    PubMed

    Lee, Jung-Hwan; Kim, Kyoung-Nam

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it. PMID:27597959

  20. The effect of hyaluronan on the motility of skin dermal fibroblasts in nanofibrous scaffolds.

    PubMed

    Qian, Yuna; Li, Linhao; Jiang, Chao; Xu, Wei; Lv, Yonggang; Zhong, Li; Cai, Kaiyong; Yang, Li

    2015-08-01

    Nanofibrous scaffolds that use the native extracellular matrix are promising developments in skin tissue regeneration because they provide the proper environment for the adhesion, migration and growth of skin dermal fibroblasts, important during wound healing. In this study, we focus on hyaluronan as a native ECM that regulates cellular motility in nanofibrous scaffolds. PCL/HA nanofibrous scaffolds were generated by electrospinning and assessed for various physicochemical properties. HA-based scaffolds significantly enhanced cell infiltration in vitro and in vivo. The observation of movements in living cells revealed that HA-based scaffolds regulated cell migration speed and direction. This phenomenon may influences by the variation in cell adhesion receptors-integrin β1, and vinculin formation and distribution. Furthermore, we confirmed that HA/CD44 interactions can activate the TGF-β/MMP-2 signaling pathway that promotes cell motility. These findings suggest HA functions in the cell motility of nanofibrous scaffolds and have potential implications for the use of HA-based scaffolds in skin tissue regeneration applications. PMID:25940528

  1. Inhibitory Effects of Terminalia catappa on UVB-Induced Photodamage in Fibroblast Cell Line

    PubMed Central

    Wen, Kuo-Ching; Shih, I-Chen; Hu, Jhe-Cyuan; Liao, Sue-Tsai; Su, Tsung-Wei; Chiang, Hsiu-Mei

    2011-01-01

    This study investigated whether Terminalia catappa L. hydrophilic extract (TCLW) prevents photoaging in human dermal fibroblasts after exposure to UVB radiation. TCLW exhibited DPPH free radical scavenging activity and protected erythrocytes against AAPH-induced hemolysis. In the gelatin digestion assay, the rates of collagenase inhibition by TCL methanol extract, TCLW, and its hydrolysates were greater than 100% at the concentration of 1 mg/mL. We found that serial dilutions of TCLW (10–500 μg/mL) inhibited collagenase activity in a dose-dependent manner (82.3% to 101.0%). However, TCLW did not significantly inhibit elastase activity. In addition, TCLW inhibited MMP-1 and MMP-9 protein expression at a concentration of 25 μg/mL and inhibited MMP-3 protein expression at a concentration of 50 μg/mL. TCLW also promoted the protein expression of type I procollagen. We also found that TCLW attenuated the expression of MMP-1, -3, and -9 by inhibiting the phosphorylation of ERK, JNK, and p38. These findings suggest that TCLW increases the production of type I procollagen by inhibiting the activity of MMP-1, -3 and -9, and, therefore, has potential use in anti-aging cosmetics. PMID:20981325

  2. Effects of Activin in Embryoid Bodies Expressing Fibroblast Growth Factor 5.

    PubMed

    Shirouzu, Yasumasa; Yanai, Goichi; Yang, Kai-Chiang; Sumi, Shoichiro

    2016-06-01

    Nodal/activin signaling is indispensable for embryonic development. We examined what activin does to the embryoid bodies (EBs) produced from mouse embryonic stem cells (mESCs) expressing an epiblast marker. The EBs were produced by culturing mESCs by the hanging drop method for 24 hours. The resulting EBs were transferred onto gelatin-coated dishes and allowed to further differentiate. The 24-hour EBs showed a stronger expression of fibroblast growth factor (FGF)5 and Brachyury (specific to the epiblast) in comparison with mESCs. Treating the transferred EBs with activin A maintained transcript levels of FGF5 and Oct4, while inhibiting definitive endoderm differentiation. The activin A treatment reversed the endoderm differentiation induced by retinoic acid (RA), while the inhibition of nodal/activin signaling promoted RA-induced endoderm differentiation. Inhibition of nodal/activin signaling in EBs, including epiblast-like cells, promotes differentiation into the endoderm, facilitating the transition from the pluripotent state to specification of the endoderm. PMID:27253628

  3. Inhibitory Effects of Terminalia catappa on UVB-Induced Photodamage in Fibroblast Cell Line.

    PubMed

    Wen, Kuo-Ching; Shih, I-Chen; Hu, Jhe-Cyuan; Liao, Sue-Tsai; Su, Tsung-Wei; Chiang, Hsiu-Mei

    2011-01-01

    This study investigated whether Terminalia catappa L. hydrophilic extract (TCLW) prevents photoaging in human dermal fibroblasts after exposure to UVB radiation. TCLW exhibited DPPH free radical scavenging activity and protected erythrocytes against AAPH-induced hemolysis. In the gelatin digestion assay, the rates of collagenase inhibition by TCL methanol extract, TCLW, and its hydrolysates were greater than 100% at the concentration of 1 mg/mL. We found that serial dilutions of TCLW (10-500 μg/mL) inhibited collagenase activity in a dose-dependent manner (82.3% to 101.0%). However, TCLW did not significantly inhibit elastase activity. In addition, TCLW inhibited MMP-1 and MMP-9 protein expression at a concentration of 25 μg/mL and inhibited MMP-3 protein expression at a concentration of 50 μg/mL. TCLW also promoted the protein expression of type I procollagen. We also found that TCLW attenuated the expression of MMP-1, -3, and -9 by inhibiting the phosphorylation of ERK, JNK, and p38. These findings suggest that TCLW increases the production of type I procollagen by inhibiting the activity of MMP-1, -3 and -9, and, therefore, has potential use in anti-aging cosmetics. PMID:20981325

  4. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  5. Quercitrin for periodontal regeneration: effects on human gingival fibroblasts and mesenchymal stem cells

    PubMed Central

    Gómez-Florit, Manuel; Monjo, Marta; Ramis, Joana M.

    2015-01-01

    Periodontal disease (PD) is the result of an infection and chronic inflammation of the gingiva that may lead to its destruction and, in severe cases, alveolar bone and tooth loss. The ultimate goal of periodontal treatment is to achieve periodontal soft and hard tissues regeneration. We previously selected quercitrin, a catechol-containing flavonoid, as a potential agent for periodontal applications. In this study, we tested the ability of quercitrin to alter biomarker production involved in periodontal regeneration on primary human gingival fibroblasts (hGF) and primary human mesenchymal stem cells (hMSC) cultured under basal and inflammatory conditions. To mimic PD inflammatory status, interleukin-1 beta (IL-1β) was used. The expression of different genes related to inflammation and extracellular matrix were evaluated and prostaglandin E2 (PGE2) production was quantified in hGFs; alkaline phosphatase (ALP) activity and calcium content were analysed in hMSCs. Quercitrin decreased the release of the inflammatory mediator PGE2 and partially re-established the impaired collagen metabolism induced by IL-1β treatment in hGFs. Quercitrin also increased ALP activity and mineralization in hMSCs, thus, it increased hMSCs differentiation towards the osteoblastic lineage. These findings suggest quercitrin as a novel bioactive molecule with application to enhance both soft and hard tissue regeneration of the periodontium. PMID:26558438

  6. [Active ingredients in rhubarb with anti-proliferative effects on scar fibroblasts].

    PubMed

    Wang, Qian; Zhang, Nan-Nan; Li, Hong-Yan; Jiang, Min; Gao, Jie; Bai, Gang

    2012-12-01

    This study is to explore the active ingredients of traditional Chinese medicine rhubarb with antiproliferative activity on hypertrophic scar fibroblasts (HSF). Rhubarb was extracted with Soxhlet extraction method by different polar solvents. MTS method was used to screen rhubarb solvent extracts (25 microg x mL(-1)) with anti-proliferative activity on HSF, and flow cytometry was used to detect their influences on cell cycle. Then, the active ingredients were analyzed by HPLC. The components with high activity were identified by UPLC-Q/TOF and verified by HE staining. The results showed that the ethyl acetate extract of rhubarb had higher anti-proliferative activity (P < 0.01), increased significantly the proportion of cells in G0/G1 phase (P < 0.01), and reduced the proliferation index (PI) (P < 0.01). The main active ingredients were anthraquinones. The results of confirming experiment showed that emodin, rhein and gallic acid could inhibit cell proliferation in a dose-dependent manner. In conclusion, the ethyl acetate extract of rhubarb showed anti-proliferative activity on HSF, and the anti-proliferative ingredients might be anthraquinones. PMID:23460967

  7. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    PubMed Central

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  8. The Effects of Chronological Age on the Cellular Mechanics of Human Dermal Fibroblasts

    NASA Astrophysics Data System (ADS)

    Pan, Z.; Hung, V.; Kambhampati, S.; Ge, S. R.; Rafailovich, M.; Ghosh, K.; Clark, R.; Liu, Y. J.; Nakamura, T.; Shu, X. Z.; Prestwich, G.

    2006-03-01

    It is often observed that older people display diminished wound healing abilities. Understanding of this phenomenon is important for many in vivo applications of tissue engineering. In this study, the cell mechanics of dermal fibroblasts from 25, 40 and 84 years old female subjects were compared. These cells were cultured on functionalized hyaluronic acid hydrogel substrates which emulated physiological conditions in dermal tissue. The deformation of the substrate caused by cellular traction forces was detected by tracing the displacement of fluorescent beads embedded in the substrate using Digital Image Speckle Correlation. Then cellular traction forces were quantitatively determined by Finite Element Method in a linear elastic model with a high spatial resolution. These results were correlated with auxiliary measurements of substrate modulus, cell modulus and migration. We found that with increasing age, the magnitude of the cellular traction forces diminished. Similarly, the ability of the cells to adapt to changes in the mechanical properties of their environment and migrate was also impaired. The interrelationship between these factors and wound healing will be discussed. This work is supported by NSF- MRSEC program.

  9. The effect of ursolic and oleanolic acids on human skin fibroblast cells.

    PubMed

    Wójciak-Kosior, Magdalena; Paduch, Roman; Matysik-Woźniak, Anna; Niedziela, Piotr; Donica, Helena

    2011-01-01

    In this article, we look at how ursolic and oleanolic acids can be used for the purpose of quality control of natural products used in dermatocosmetology as well as of various other therapeutic preparations. Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenes and they are constituents of many medicinal herbs. In this study, we analyzed the cytotoxic and anti-proliferative activity of OA and UA against normal human skin fibroblasts (HSF). Additionally, the scavenging activity of free radicals of both acids was analyzed. The sensitivity of cells to OA and UA activity was determined using a standard spectrophotometric (MTT) assay. The free radical scavenging activity of OA and UA was measured using the DPPH• test. The F-actin cytoskeletal proteins organization was analyzed using TRITC-phalloidine fluorescent staining. The cytotoxic activity of the analyzed acids was determined using Neutral Red (NR) uptake assay. Of the two isomeric compounds, UA showed a higher cytotoxic activity against HSF cells than did OA. Our investigations showed that OA, in view of its non-toxic nature, may be used as a supplementary factor for dermal preparations. PMID:22252762

  10. Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ.

    PubMed Central

    Boon, M R; Zammit, V A

    1988-01-01

    1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo. PMID:3281653

  11. Biophysical Studies of Nanosecond Pulsed Electric Field Induced Cell Membrane Permeabilization

    NASA Astrophysics Data System (ADS)

    Wu, Yu-Hsuan

    Nanosecond megavolts-per-meter pulsed electric field (nsPEF) offers a non-invasive manipulation of intracellular organelles and functions of biological cells. Accordingly, nsPEF is a potential technique for biophysical research and cancer therapy, and is of growing interest. Although, the application of nsPEF has shown electroperturbation on cell plasma membranes and intracellular membranes as well, the mechanisms underlying the electropermeabilization are still not clear. In this thesis, we systematically study nsPEFs (5 and 30 ns) induced membrane permeability change in biological cell in-vitro with different pulse parameters. In Chapter 3, we investigate the nsPEF-induced intracellular membrane permeabilization of mitochondria which play key roles in activating apoptosis in mammalian cells. The results show the evidences of nsPEF-induced membrane permeability increase in mitochondria, and suggest that nsPEF is a potential technology for cancer cell ablation without delivery of drug or gene into cells. In Chapter 2, 4 and 6, we study the properties of nsPEF-induced plasma membrane permeabilization. In the beginning, the change of plasma membrane permeability is studied by uptake of YO-PRO-1 and propidium iodide, fluorescent dyes specifically used as indicators of plasma membrane permeabilization. However, the detection is limited by the fluorescent emission efficiency and detector capability. To increase the detection sensitivity, we later develop a method based on cell volume change due to regulation of osmotic balance that causes water and small ions transport through plasma membrane. We find that even a single 10 MV/m pulse of 5 ns duration produces measureable cell swelling. The results demonstrate that cell swelling is susceptible to nsPEF and can detect membrane permeabilization more easily and precisely than fluorescent dyes. We compare the effects of different pulse parameters (pulse duration, pulse number, electric field amplitude and pulse repetition

  12. Changes in transcription profile and cytoskeleton morphology in pelvic ligament fibroblasts in response to stretch: the effects of estradiol and levormeloxifene.

    PubMed

    Ewies, Ayman A A; Elshafie, Mona; Li, Jin; Stanley, Adrian; Thompson, John; Styles, Jerry; White, Ian; Al-Azzawi, Farook

    2008-02-01

    Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of the molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered as a result of mechanical stretch. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static. Out of 34 mechano-responsive genes identified (P < 0.05), four were coding for regulation of actin cytoskeleton remodelling, and its interaction with the extracellular matrix proteins; these are phosphatidyl inositol-4-phosphate 5-kinase (PIP5K1C), the human signal-induced proliferation associated gene-1 (SIPA-1), TNFRSF1A-associated via death domain (TRADD) and deoxyribonuclease 1-like 1 (DNase 1-L1). The transcriptosomal changes led us to investigate the phenotypic consequences of stretch, levormeloxifene and estradiol (E(2)) on the cytoskeleton of cultured fibroblasts. The percentage of cells with abnormal F-actin configuration was significantly higher in fibroblasts subjected to stretch compared with the static model (P < 0.0001). Levormeloxifene caused similar significant alterations in actin morphology of the static fibroblasts. The use of E(2) did not reverse the process or protect the cells from the effect of stretch, but significantly increased the rate of fibroblast proliferation, suggestive of a role in healing process. Mechanical stretch and/or levormeloxifene disturb the fibroblasts ability to maintain the cytoskeleton architecture and we speculate that they may disrupt ligamentous integrity and result in clinical prolapse. PMID:18184756

  13. Involvement of B2 Receptor in Bradykinin-Induced Proliferation and Proinflammatory Effects in Human Nasal Mucosa-Derived Fibroblasts Isolated from Chronic Rhinosinusitis Patients

    PubMed Central

    Tsai, Yih-Jeng; Hao, Sheng-Po; Chen, Chih-Li; Lin, Brian J.; Wu, Wen-Bin

    2015-01-01

    Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa either accompanied by polyp formation (CRSwNP) or without polyps (CRSsNP). CRSsNP accounts for the majority of CRS cases and is characterized by fibrosis and neutrophilic inflammation. However, the pathogenesis of CRS, especially CRSsNP, remains unclear. Immunohistochemistry of CRSsNP specimens in the present study showed that the submucosa, perivascular areas, and the mucous glands were abundant in fibroblasts. Therefore, we investigated the effects bradykinin (BK), an autacoid known to participate in inflammation, on human CRSsNP nasal mucosa-derived fibroblasts (NMDFs). BK increased CXCL1 and -8 secretion and mRNA expression with EC50 ranging from 0.15~0.35 μM. Moreover, BK enhanced cell proliferation and upregulated the expressions of proinflammatory molecules, including cell adhesion molecules (CAMs) and cyclooxygenase (COX)-1 and -2. These functionally caused an increase in monocyte adhesion to fibroblast monolayer. Using pharmacological intervention and BKR siRNA knockdown, we demonstrated that the BK-induced CXCL chemokine release, cell proliferation and COX and CAM expressions were mainly through the B2 receptor (B2R). Accordingly, the B2R was preferentially expressed in the NMDFs than B1R. The B2R was highly expressed in the CRSsNP than the control specimens, while the B1R and kininogen (KNG)/BK expression slightly increased in the CRSsNP mucosa. Collectively, we report here for the first time that fibroblasts, KNG/BK, and BKRs are overexpressed in CRSsNP mucosa and BK upregulates chemokine expression, proliferation, and proinflammatory molecule expression in NMDFs via B2R activation, which lead to a functional increase in monocyte-fibroblast interaction. Our findings reveal a critical role of fibroblast, KNG/BK, and BKRs in the development of CRSsNP. PMID:25970620

  14. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling. PMID:26632599

  15. The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome.

    PubMed

    Kanabus, Marta; Fassone, Elisa; Hughes, Sean David; Bilooei, Sara Farahi; Rutherford, Tricia; Donnell, Maura O'; Heales, Simon J R; Rahman, Shamima

    2016-05-01

    There is growing interest in the use of the ketogenic diet (KD) to treat inherited metabolic diseases including mitochondrial disorders. However, neither the mechanism whereby the diet may be working, nor if it could benefit all patients with mitochondrial disease, is known. This study focusses on decanoic acid (C10), a component of the medium chain triglyceride KD, and a ligand for the nuclear receptor PPAR-γ known to be involved in mitochondrial biogenesis. The effects of C10 were investigated in primary fibroblasts from a cohort of patients with Leigh syndrome (LS) caused by nuclear-encoded defects of respiratory chain complex I, using mitochondrial respiratory chain enzyme assays, gene expression microarray, qPCR and flow cytometry. Treatment with C10 increased citrate synthase activity, a marker of cellular mitochondrial content, in 50 % of fibroblasts obtained from individuals diagnosed with LS in a PPAR-γ-mediated manner. Gene expression analysis and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst downregulating genes involved in glucose metabolism (PDK3, PDK4). PCK2, involved in blocking glucose metabolism, was upregulated, as was CAT, encoding catalase. Moreover, treatment with C10 also decreased oxidative stress in complex I deficient (rotenone treated) cells. However, since not all cells from subjects with LS appeared to respond to C10, prior cellular testing in vitro could be employed as a means for selecting individuals for subsequent clinical studies involving C10 preparations. PMID:27080638

  16. Detection of electroporation-induced membrane permeabilization states in the brain using diffusion-weighted MRI

    PubMed Central

    Mahmood, Faisal; Hansen, Rasmus H.; Agerholm-Larsen, Birgit; Gissel, Hanne; Ibsen, Per

    2015-01-01

    Background Tissue permeabilization by electroporation (EP) is a promising technique to treat certain cancers. Non-invasive methods for verification of induced permeabilization are important, especially in deep-seated cancers. In this study we evaluated diffusion-weighted magnetic resonance imaging (DW-MRI) as a quantitative method for detecting EP-induced membrane permeabilization of brain tissue using a rat brain model. Material and methods Fifty-four anesthetized Sprague-Dawley male rats were electroporated in the right hemisphere, using different voltage levels to induce no permeabilization (NP), transient membrane permeabilization (TMP), and permanent membrane permeabilization (PMP), respectively. DW-MRI was acquired 5 minutes, 2 hours, 24 hours and 48 hours after EP. Histology was performed for validation of the permeabilization states. Tissue content of water, Na+, K+, Ca2+, and extracellular volume were determined. The Kruskal-Wallis test was used to compare the DW-MRI parameters, apparent diffusion coefficient (ADC) and kurtosis, at different voltage levels. The two-sample Mann- Whitney test with Holm's Bonferroni correction was used to identify pairs of significantly different groups. The study was approved by the Danish Animal Experiments Inspectorate. Results and conclusion Results showed significant difference in the ADC between TMP and PMP at 2 hours (p < 0.001) and 24 hours (p < 0.05) after EP. Kurtosis was significantly increased both at TMP (p < 0.05) and PMP (p < 0.001) 5 minutes after EP, compared to NP. Kurtosis was also significantly higher at 24 hours (p < 0.05) and 48 hours (p < 0.05) at PMP compared to NP. Physiological parameters indicated correlation with the permeabilization states, supporting the DW-MRI findings. We conclude that DW-MRI is capable of detecting EP-induced permeabilization of brain tissue and to some extent of differentiating NP, TMP and PMP using appropriate scan timing. PMID:25591820

  17. Effect of diode low-level lasers on fibroblasts derived from human periodontal tissue: a systematic review of in vitro studies.

    PubMed

    Ren, Chong; McGrath, Colman; Jin, Lijian; Zhang, Chengfei; Yang, Yanqi

    2016-09-01

    This study aimed to systematically assess the parameter-specific effects of the diode low-level laser on human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPDLFs). An extensive search was performed in major electronic databases including PubMed (1997), EMBASE (1947) and Web of Science (1956) and supplemented by hand search of reference lists and relevant laser journals for cell culture studies investigating the effect of diode low-level lasers on HGFs and HPDLFs published from January 1995 to December 2015. A total of 21 studies were included after screening 324 independent records, amongst which eight targeted HPDLFs and 13 focussed on HGFs. The diode low-level laser showed positive effects on promoting fibroblast proliferation and osteogenic differentiation and modulating cellular inflammation via changes in gene expression and the release of growth factors, bone-remodelling markers or inflammatory mediators in a parameter-dependent manner. Repeated irradiations with wavelengths in the red and near-infrared range and at an energy density below 16 J/cm(2) elicited favourable responses. However, considerable variations and weaknesses in the study designs and laser protocols limited the interstudy comparison and clinical transition. Current evidence showed that diode low-level lasers with adequate parameters stimulated the proliferation and modulated the inflammation of fibroblasts derived from human periodontal tissue. However, further in vitro studies with better designs and more appropriate study models and laser parameters are anticipated to provide sound evidence for clinical studies and practice. PMID:27422104

  18. Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes

    PubMed Central

    2012-01-01

    Background Fibroblast growth factor receptor 3 (FGFR3) inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3) plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT) and RNA component of telomerase (TR), and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response. Results Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p < 0.05). Chondrocytes treated with T3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p < 0.05); TERT and TR were not significantly reduced. The action of T3 at the growth plate may be partially mediated through the FGFR3 pathway. Conclusions The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation. PMID:23216972

  19. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    PubMed

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-01

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development. PMID:27402846

  20. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

    PubMed

    Rogers, Carlyle; Davis, Barbara; Neufer, P Darrell; Murphy, Michael P; Anderson, Ethan J; Robidoux, Jacques

    2014-02-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels. PMID:24269897

  1. Effective treatment of steatosis and steatohepatitis by fibroblast growth factor 1 in mouse models of nonalcoholic fatty liver disease

    PubMed Central

    Liu, Weilin; Struik, Dicky; Nies, Vera J. M.; Jurdzinski, Angelika; Harkema, Liesbeth; de Bruin, Alain; Verkade, Henkjan J.; Downes, Michael; Evans, Ronald M.; van Zutphen, Tim; Jonker, Johan W.

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder and is strongly associated with obesity and type 2 diabetes. Currently, there is no approved pharmacological treatment for this disease, but improvement of insulin resistance using peroxisome proliferator-activated receptor-γ (PPARγ) agonists, such as thiazolidinediones (TZDs), has been shown to reduce steatosis and steatohepatitis effectively and to improve liver function in patients with obesity-related NAFLD. However, this approach is limited by adverse effects of TZDs. Recently, we have identified fibroblast growth factor 1 (FGF1) as a target of nuclear receptor PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling. Because FGF1 is situated downstream of PPARγ, it is likely that therapeutic targeting of the FGF1 pathway will eliminate some of the serious adverse effects associated with TZDs. Here we show that pharmacological administration of recombinant FGF1 (rFGF1) effectively improves hepatic inflammation and damage in leptin-deficient ob/ob mice and in choline-deficient mice, two etiologically different models of NAFLD. Hepatic steatosis was effectively reduced only in ob/ob mice, suggesting that rFGF1 stimulates hepatic lipid catabolism. Potentially adverse effects such as fibrosis or proliferation were not observed in these models. Because the anti-inflammatory effects were observed in both the presence and absence of the antisteatotic effects, our findings further suggest that the anti-inflammatory property of rFGF1 is independent of its effect on lipid catabolism. Our current findings indicate that, in addition to its potent glucose-lowering and insulin-sensitizing effects, rFGF1 could be therapeutically effective in the treatment of NAFLD. PMID:26858440

  2. Effective treatment of steatosis and steatohepatitis by fibroblast growth factor 1 in mouse models of nonalcoholic fatty liver disease.

    PubMed

    Liu, Weilin; Struik, Dicky; Nies, Vera J M; Jurdzinski, Angelika; Harkema, Liesbeth; de Bruin, Alain; Verkade, Henkjan J; Downes, Michael; Evans, Ronald M; van Zutphen, Tim; Jonker, Johan W

    2016-02-23

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder and is strongly associated with obesity and type 2 diabetes. Currently, there is no approved pharmacological treatment for this disease, but improvement of insulin resistance using peroxisome proliferator-activated receptor-γ (PPARγ) agonists, such as thiazolidinediones (TZDs), has been shown to reduce steatosis and steatohepatitis effectively and to improve liver function in patients with obesity-related NAFLD. However, this approach is limited by adverse effects of TZDs. Recently, we have identified fibroblast growth factor 1 (FGF1) as a target of nuclear receptor PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling. Because FGF1 is situated downstream of PPARγ, it is likely that therapeutic targeting of the FGF1 pathway will eliminate some of the serious adverse effects associated with TZDs. Here we show that pharmacological administration of recombinant FGF1 (rFGF1) effectively improves hepatic inflammation and damage in leptin-deficient ob/ob mice and in choline-deficient mice, two etiologically different models of NAFLD. Hepatic steatosis was effectively reduced only in ob/ob mice, suggesting that rFGF1 stimulates hepatic lipid catabolism. Potentially adverse effects such as fibrosis or proliferation were not observed in these models. Because the anti-inflammatory effects were observed in both the presence and absence of the antisteatotic effects, our findings further suggest that the anti-inflammatory property of rFGF1 is independent of its effect on lipid catabolism. Our current findings indicate that, in addition to its potent glucose-lowering and insulin-sensitizing effects, rFGF1 could be therapeutically effective in the treatment of NAFLD. PMID:26858440

  3. [Characteristics of the effect of exometabolites of the cysticerci of the cat tapeworm Hydatigera taeniaeformis on fibroblasts in organ cultures].

    PubMed

    Adoeva, E I

    1987-11-01

    The influence of the cestode H. taeniaeformis cysticercus exometabolites on the fibroblasts of their capsule in organ culture was studied. Half of explants were cultured in the presence of cestodes (the ratio between the parasite body weight and the cultural medium volume being 1:10 g/ml). After 2-30 days of cultivation the explants were examined by histological, histoautoradiographic and electron microscopic methods. The influence of cysticercus exometabolites on the culture resembled that of the growth-stimulating factors. The young, actively proliferating fibroblasts were mainly observed in the explants of the capsules cultured in the presence of cysticerci. Among the mature cells, fibroblasts with the predominance of the fibroclastic function were noticed rather than with the function of collagen biosynthesis. It is suggested that the change in the differentiation ways of fibroblasts may be due to some biologically active substrates of the cysticercus to be directed on slowing down the maturation of the connective tissue capsule. PMID:3438936

  4. Effect of Extracts of Terminalia chebula on Proliferation of Keratinocytes and Fibroblasts Cells: An Alternative Approach for Wound Healing

    PubMed Central

    Choi, Soon Mo; Zo, Sun Mi; Painuli, Rakesh Mohan; Kwon, Sung Won; Han, Sung Soo

    2014-01-01

    Terminalia chebula is one of the traditional medicines used in the treatment of many diseases. In the present work, different concentrations of various organic and aqueous extracts (solvent-free) of T. chebula were tested on fibroblast (L929) and keratinocytes cells to evaluate its biocompatible concentration by using MTT and live-dead viability/cytotoxic assay. These extracts were found to be effective in decreasing the ammonia accumulation in the media, thereby reducing its toxic effect on cells. DPPH assay further confirmed the free-radical scavenging ability of the extracts which increased with the increase in concentration of each extract. Cell proliferation/apoptosis, cytoskeletal structure, and ECM production were further evaluated by live-dead assay and phalloidin/cytokeratin staining, respectively. The cytoskeletal structure and ECM secretion of the cells treated with extracts showed higher cellular activity in comparison to control. In conclusion, we have demonstrated the effect of these extracts of T. chebula on both types of skin cells and optimized concentration in which it could be used as a bioactive component for wound healing applications by increasing cell proliferation and decreasing free-radical production without affecting the normal cellular matrix. It can also find applications in other therapeutics applications where ammonia toxicity is a limiting factor. PMID:24719644

  5. The effects of UV emission from compact fluorescent light exposure on human dermal fibroblasts and keratinocytes in vitro.

    PubMed

    Mironava, Tatsiana; Hadjiargyrou, Michael; Simon, Marcia; Rafailovich, Miriam H

    2012-01-01

    Compact fluorescent light (CFL) bulbs can provide the same amount of lumens as incandescent light bulbs, using one quarter of the energy. Recently, CFL exposure was found to exacerbate existing skin conditions; however, the effects of CFL exposure on healthy skin tissue have not been thoroughly investigated. In this study, we studied the effects of exposure to CFL illumination on healthy human skin tissue cells (fibroblasts and keratinocytes). Cells exposed to CFLs exhibited a decrease in the proliferation rate, a significant increase in the production of reactive oxygen species, and a decrease in their ability to contract collagen. Measurements of UV emissions from these bulbs found significant levels of UVC and UVA (mercury [Hg] emission lines), which appeared to originate from cracks in the phosphor coatings, present in all bulbs studied. The response of the cells to the CFLs was consistent with damage from UV radiation, which was further enhanced when low dosages of TiO(2) nanoparticles (NPs), normally used for UV absorption, were added prior to exposure. No effect on cells, with or without TiO(2) NPs, was observed when they were exposed to incandescent light of the same intensity. PMID:22724459

  6. In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice

    PubMed Central

    Khoo, Nooshafarin Kazemi; Shokrgozar, Mohammad Ali; Kashani, Iraj Ragerdi; Amanzadeh, Amir; Mostafavi, Ehsan; Sanati, Hassan; Habibi, Laleh; Talebi, Saeid; Abouzaripour, Morteza; Akrami, Seyed Mohammad

    2014-01-01

    Background Numerous in vitro reports suggest that Low Level Laser Therapy (LLLT) affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light (which is more useful in clinic because of its higher penetration) on secretion of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF), as important growth factors in wound healing. Methods Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT (energy density of 1 J/cm 2) using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time poly-merase chain reaction. Results Diabetic irradiated group showed significant increase in FGF (p = 0.017) expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. Conclusion These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts. PMID:24834313

  7. Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

    PubMed

    Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

    2016-09-01

    The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p < 0.05) on mean absorbance of fibroblasts cultured with 10 and 25 % PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo. PMID:27394438

  8. Effects of a fibrin-fibronectin sealing system on proliferation and type I collagen synthesis of human PDL fibroblasts in vitro.

    PubMed

    Fabris, G; Trombelli, L; Schincaglia, G P; Cavallini, R; Calura, G; del Senno, L

    1998-01-01

    Fibrin glue (FG) is an agent widely used in many surgical disciplines for achieving hemostasis and tissue adhesion. The aim of this investigation was to determine the effectiveness of a highly concentrated FG (Tissucol) on the growth and phenotypic expression of human periodontal ligament (PDL) fibroblasts. PDL fibroblast strains were established from cells scraped from PDL, and cultured in the presence and absence of FG for 48 and 72 h. Cell proliferation was studied by counting cells and mitoses, and by immunocytochemical detection of the proliferation-associated Ki-67 nuclear antigen. Type-I collagen production was assessed by radioimmunological assay of the procollagen C-terminal peptide. Results showed that FG treatment was compatible with PDL fibroblast growth and type-I collagen synthesis, although a reduced trend in cell proliferation and collagen production was found in FG-supplemented cultures compared to control cultures. We conclude that FG may represent a suitable substrate for supporting PDL fibroblast growth and function. PMID:9477014

  9. Quantitative measurement of permeabilization of living cells by terahertz attenuated total reflection

    NASA Astrophysics Data System (ADS)

    Grognot, Marianne; Gallot, Guilhem

    2015-09-01

    Using Attenuated Total Reflection imaging technique in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of epithelial cells by saponin. The terahertz signal is mostly sensitive to the intracellular protein concentration in the cells, in a very good agreement with standard bicinchoninic acid protein measurements. It opens the way to in situ real time dynamics of protein content and permeabilization in live cells.

  10. Short communication: opposing effects of lactoferrin on the proliferation of fibroblasts and epithelial cells from bovine mammary gland.

    PubMed

    Nakajima, K; Itoh, F; Nakamura, M; Kawamura, A; Yamazaki, T; Kozakai, T; Takusari, N; Ishisaki, A

    2015-02-01

    Lactoferrin is present in several physiologic fluids, including milk and colostrum. Recently, evidence has accumulated that lactoferrin acts as a regulator of cell proliferation. Lactoferrin mRNA and protein levels in bovine mammary glands are known to markedly increase after cessation of milking. To clarify the role of bovine lactoferrin (bLF) in mammary involution and remodeling during dry periods, we investigated whether bLF affects the proliferation of cultured cells derived from bovine mammary gland and examined the mechanism underlying the proliferative response to bLF. Addition of bLF to the culture medium increased the proliferation of bovine mammary stromal fibroblasts (bMSF), but decreased that of bovine mammary epithelial cells (bMEC). Proliferation was significantly increased in the bMSF treated with bLF (100μg/mL or greater) as compared with unstimulated cells. The maximal proliferative effect of bLF on bMSF occurred at 1,000μg/mL, such that the proliferation of the bLF-stimulated bMSF was approximately 2.5 times that of unstimulated cells. The bLF increased the production of proliferating cell nuclear antigen and rapid phosphorylation of the p44/p42 mitogen-activated protein kinase in bMSF, but not in bMEC. The bLF-induced proliferation and production of proliferating cell nuclear antigen in bMSF was suppressed by U0126, a specific inhibitor of mitogen-activated protein kinase. Furthermore, treatment with bLF for 24h decreased the mRNA levels of the 3 isoforms of transforming growth factor β in bMSF (16-66%) but upregulated those in bMEC (122-157%). These opposite effects of bLF on the proliferation of epithelial and fibroblast cells and their expression of transforming growth factor β may play a crucial role in bovine mammary involution and remodeling. PMID:25497822

  11. Arctiin induces an UVB protective effect in human dermal fibroblast cells through microRNA expression changes.

    PubMed

    Lee, Ghang Tai; Cha, Hwa Jun; Lee, Kwang Sik; Lee, Kun Kook; Hong, Jin Tae; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Bae, Seunghee

    2014-03-01

    Ultraviolet (UV) radiation induces severe alterations in the molecular and cellular components of normal human dermal fibroblast (NHDF) cells by disrupting many intracellular transduction cascades. Although UV responses have been well documented at the genome and proteome levels, UV protective effects have not been elucidated at these levels. The aim of the present study was to demonstrate that arctiin, a phytochemical isolated from the plant Arctium lappa, induced a protective effect against UVB radiation by changing microRNA (miRNA) expression profiles. Using flow cytometry, and water-soluble tetrazolium salt (WST-1)-based cell viability, wound healing, and DNA repair assays we showed that pretreatment with arctiin prior to UVB irradiation reduced UVB-induced apoptosis, cell migration defects, and DNA damage in NHDF cells. It was also found that arctiin‑induced UVB protection is associated with altered miRNA expression profiles. Bioinformatic analysis revealed that the deregulated miRNAs were functionally involved in mitogen-activated protein kinase (MAPK) signaling and cancer signaling pathways. The results suggest that arctiin acts as a UVB protective agent by altering specific miRNA expression in NHDF cells. PMID:24398562

  12. Chaperone therapy for GM2 gangliosidosis: effects of pyrimethamine on β-hexosaminidase activity in Sandhoff fibroblasts.

    PubMed

    Chiricozzi, Elena; Niemir, Natalia; Aureli, Massimo; Magini, Alessandro; Loberto, Nicoletta; Prinetti, Alessandro; Bassi, Rosaria; Polchi, Alice; Emiliani, Carla; Caillaud, Catherine; Sonnino, Sandro

    2014-08-01

    Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from β-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of β-hexosaminidase activity in cultured fibroblasts from Sandhoff patients. PMID:24356898

  13. Comparative in vitro study of the effectiveness of Green tea extract and common storage media on periodontal ligament fibroblast viability

    PubMed Central

    Adeli, Fahimeh; Zabihi, Ebrahim; Abedian, Zeinab; Gharekhani, Samane; Pouramir, Mahdi; Khafri, Soraya; Ghasempour, Maryam

    2016-01-01

    Objective: Green tea extract (GTE) was shown to be effective in preserving periodontal ligament fibroblasts (PDLFs) of avulsed teeth. This study aimed at determining the potential of GTE in preserving the viability of PDLFs comparing with different storage media. Materials and Methods: Periodontal ligament cells were obtained from freshly extracted healthy impacted third molars and cultured in Dulbecco's Modified Eagle Medium (DMEM). Cell viability was determined by storing the cells in seven media; DMEM, tap water, Hank's balanced salt solution (HBSS), whole milk, hypotonic sucrose solution, GTE, and GTE + sucrose for 1, 2, 4, and 24 h at 37°C using tetrazolium salt-based colorimetric (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay. Statistical analysis was performed by one-way analysis of variance and post hoc tests. Results: GTE showed significantly higher protective effect than HBSS at 2, 4, and 24 h (P = 0.009, P = 0.02, P = 0.016), DMED at 2 h (P = 0.003), and milk at 4 h (P = 0.039). Conclusion: Although with undesirable osmolality and pH, GTE had a good ability in preserving the PDLFs comparing with other studied media. PMID:27403063

  14. Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

    PubMed Central

    Zhang, L.; Ding, Y.; Rao, G.Z.; Miao, D.

    2016-01-01

    The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease. PMID:27074164

  15. Dependence of the bystander effect for micronucleus formation on dose of heavy-ion radiation in normal human fibroblasts.

    PubMed

    Matsumoto, Yoshitaka; Hamada, Nobuyuki; Aoki-Nakano, Mizuho; Funayama, Tomoo; Sakashita, Tetsuya; Wada, Seiichi; Kakizaki, Takehiko; Kobayashi, Yasuhiko; Furusawa, Yoshiya

    2015-09-01

    Ionising radiation-induced bystander effects are well recognised, but its dependence on dose or linear energy transfer (LET) is still a matter of debate. To test this, 49 sites in confluent cultures of AG01522D normal human fibroblasts were targeted with microbeams of carbon (103 keV µm(-1)), neon (375 keV µm(-1)) and argon ions (1260 keV µm(-1)) and evaluated for the bystander-induced formation of micronucleus that is a kind of a chromosome aberration. Targeted exposure to neon and argon ions significantly increased the micronucleus frequency in bystander cells to the similar extent irrespective of the particle numbers per site of 1-6. In contrast, the bystander micronucleus frequency increased with increasing the number of carbon-ion particles in a range between 1 and 3 particles per site and was similar in a range between 3 and 8 particles per site. These results suggest that the bystander effect of heavy ions for micronucleus formation depends on dose. PMID:26242975

  16. Effects of Narrow-band IR-A and of Water-Filtered Infrared A on Fibroblasts.

    PubMed

    Knels, Lilla; Valtink, Monika; Piazena, Helmut; de la Vega Marin, Jamlec; Gommel, Kerstin; Lupp, Amelie; Roehlecke, Cora; Mehner, Mirko; Funk, Richard H W

    2016-05-01

    Exposures of the skin with electromagnetic radiation of wavelengths between 670 nm and 1400 nm are often used as a general treatment to improve wound healing and reduce pain, for example, in chronic diabetic skin lesions. We investigated the effects of water-filtered infrared A (wIRA) and of narrow-band IR-A provided by a light-emitting diode LED (LED-IR-A) irradiation in vitro on 3T3 fibroblast cultures under defined conditions with and without glyoxal administration. Glyoxal triggers the formation of advanced glycation end products, thereby mimicking a diabetic metabolic state. Cell viability and apoptotic changes were determined by flow cytometry after vital staining with Annexin V, YO-PRO-1 and propidium iodide (PI), and by SubG1 assay. Mitochondrial function and oxidative stress were examined by vital staining for radical production, mitochondrial membrane potential (MMP) and the ratio of reduced-to-oxidized glutathione (GSH/GSSG). The metabolic state was monitored by a resazurin conversion assay. The numbers of apoptotic cells were reduced in cultures irradiated with wIRA or LED-IR-A. More mitochondria showed a well-polarized MMP after wIRA irradiation in glyoxal damaged cells. LED-IR-A treatment specifically restored the GSH/GSSG ratio. The immediate positive effects of wIRA and LED-IR-A observed in living cells, particularly on mitochondria, reflect the therapeutic benefits of wIRA and LED-IR-A. PMID:26876482

  17. A Pilot Study of the Photoprotective Effects of Strawberry-Based Cosmetic Formulations on Human Dermal Fibroblasts

    PubMed Central

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Yuliett; Afrin, Sadia; Alvarez-Suarez, José Miguel; Gonzàlez-Paramàs, Ana Maria; Santos-Buelga, Celestino; Bompadre, Stefano; Quiles, José Luis; Mezzetti, Bruno; Giampieri, Francesca

    2015-01-01

    Strawberry polyphenols have been extensively studied over the last two decades for their beneficial properties. Recently, their possible use in ameliorating skin conditions has also been proposed; however, their role in preventing UVA-induced damage in cosmetic formulation has not yet been investigated. Skin is constantly exposed to several environmental stressors, such as UVA radiation, that induce oxidative stress, inflammation and cell death via the production of reactive oxygen species (ROS). In the present study, we assessed the potential photoprotective capacity of different strawberry-based formulations, enriched with nanoparticles of Coenzyme Q10 and with sun protection factor 10 (SPF10), in human dermal fibroblasts (HuDe) exposed to UVA radiation. We confirmed that strawberries are a very rich source of polyphenols, anthocyanins and vitamins, and possess high total antioxidant capacity. We also showed that strawberry extracts (25 μg/mL–1 mg/mL) exert a noticeable photoprotection in HuDe, increasing cell viability in a dose-dependent way, and that these effects are potentiated by the presence of CoQ10red (100 μg/mL). We have demonstrated for the first time that the topical use of strawberry extract may provide good photoprotection, even if more in-depth studies are strongly encouraged in order to evaluate the cellular and molecular effects of strawberry protection. PMID:26247940

  18. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    PubMed Central

    Murase, Hiromi; Shimazawa, Masamitsu; Kakino, Mamoru; Ichihara, Kenji; Tsuruma, Kazuhiro; Hara, Hideaki

    2013-01-01

    Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation. PMID:24416064

  19. Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

    PubMed

    Mytych, Jennifer; Wnuk, Maciej; Rattan, Suresh I S

    2016-04-01

    Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells. PMID:26814705

  20. (Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture

    SciTech Connect

    Meeuwen, J.A. van . E-mail: J.A.vanMeeuwen@iras.uu.nl; Korthagen, N.; Jong, P.C. de; Piersma, A.H.; Berg, M. van den

    2007-06-15

    In the public opinion, phytochemicals (PCs) present in the human diet are often considered beneficial (e.g. by preventing breast cancer). Two possible mechanisms that could modulate tumor growth are via interaction with the estrogen receptor (ER) and inhibition of aromatase (CYP19). Multiple in vitro studies confirmed that these compounds act estrogenic, thus potentially induce tumor growth, as well as aromatase inhibitory, thus potentially reduce tumor growth. It is thought that in the in vivo situation breast epithelial (tumor) cells communicate with surrounding connective tissue by means of cytokines, prostaglandins and estradiol forming a complex feedback mechanism. Recently our laboratory developed an in vitro co-culture model of healthy mammary fibroblasts and MCF-7 cells that (at least partly) simulated this feedback mechanism (M. Heneweer et al., TAAP vol. 202(1): 50-58, 2005). In the present study biochanin A, chrysin, naringenin, apigenin, genistein and quercetin were studied for their estrogenic properties (cell proliferation, pS2 mRNA) and aromatase inhibition in MCF-7 breast tumor cells, healthy mammary fibroblasts and their co-culture. The proliferative potency of these compounds in the MCF-7 cells derived from their EC{sub 50}s decreased in the following order: estadiol (4*10{sup -3} nM) > biochanin A (9 nM) > genistein (32 nM) > testosterone (46 nM) > naringenin (287 nM) > apigenin (440 nM) > chrysin (4 {mu}M). The potency to inhibit aromatase derived from their IC{sub 50}s decreased in the following order: chrysin (1.5 {mu}M) > naringenin (2.2 {mu}M) > genistein (3.6 {mu}M) > apigenin (4.1 {mu}M) > biochanin A (25 {mu}M) > quercetin (30 {mu}M). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1-10 {mu}M). Results from co-cultures did not elucidate the dominant effect of these compounds. MCF-7 cell proliferation occurs at concentrations that are not uncommon in blood

  1. Melanogenesis inhibitory and fibroblast proliferation accelerating effects of noroleanane- and oleanane-type triterpene oligoglycosides from the flower buds of Camellia japonica.

    PubMed

    Nakamura, Seikou; Moriura, Toshitsugu; Park, Sunmi; Fujimoto, Katsuyoshi; Matsumoto, Takahiro; Ohta, Tomoe; Matsuda, Hisashi; Yoshikawa, Masayuki

    2012-08-24

    A 28-noroleanane-type triterpene oligoglycoside, camellioside E (4), an oleanane-type triterpene oligoglycoside, camellioside F (5), and the known compounds camelliosides A (1) and D (3) were isolated from a 50% EtOH extract of Camellia japonica flower buds from Korea. The principal constituents (1 and 5) significantly inhibited melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. Camellioside B (2), a major constituent of C. japonica grown in Japan, showed potent inhibition of melanogenesis [95.0 ± 1.0% (p < 0.01) at 20 μM]. The inhibitory effects of 1, 2, and 5 were stronger than that of the reference compound, arbutin. We believe the melanogenesis inhibitory effects of 2 and 5 are partly related to the proliferation inhibitory effects in B16 melanoma 4A5 cells. Conversely, camelliosides tended to enhance proliferation in normal human neonatal skin fibroblasts. Interestingly, camellioside B (2) significantly accelerated fibroblast proliferation. This biological selectivity could make camellioside B useful for treating skin disorders. Herein, we report the first scientific investigation of a triterpene that displays an inhibitory effect on melanogenesis, but that also has an enhancing effect on fibroblast proliferation. PMID:22834923

  2. Evaluation of cytotoxic effects of Anbarnesa on fibroblast L929: Can it be used as a mouthwash?

    PubMed Central

    Shafiee, Hassan Ali; Motamedi, Mohammad Hosein Kalantar; Mina, Morteza; Taheri, Jamileh Beigom; Azimi, Somayyeh; Joharchi, Khojasteh; Yadegari, Zahra; Rasouli, Hamid Reza

    2014-01-01

    Aims: In Iranian traditional medicine Anbarnesa (derived from smoke from burning female donkey's stool) has been used to treat ulcers and inflammatory conditions like stomatitis and ear infections (otitis). We assess the properties of Anbarnesa as an alternative mouthwash. Materials and Methods: In this experimental study, Anbarnesa smoke was analyzed using aGC-mass device. The smoke collected was dissolved at different densities in propylene glycol and incubated in Dulbecco's modified Eagle's medium in direct contact with fibroblast cells. Assessment of cytotoxicity was done at 1, 24 and 72 h. Cell viability was measured by methyl thiazolyl tetrazolium test, and ELISA Reader machine was used to read the results. Data were analyzed using one-way ANOVA test. Results: The findings of this study showed Anbarnesa was nontoxic in 1/64, 1/128 and 1/256 dilutions. In 1/32 dilution, toxicity was seen after 72 h. In dilutions, 1/8 and 1/16 toxicity were seen in the 1st h. Conclusion: According to the initial results of Anbarnesa may be used as an alternative mouthwash with fewer side-effects for plaque control and prevention of periodontal disease. PMID:25593399

  3. A comparative study on the possible cytotoxic effects of different nanostructured lipid carrier (NLC) compositions in human dermal fibroblasts.

    PubMed

    Brugè, Francesca; Damiani, Elisabetta; Marcheggiani, Fabio; Offerta, Alessia; Puglia, Carmelo; Tiano, Luca

    2015-11-30

    Nanostructured lipid carriers (NLC) are widely used for topical delivery of active ingredients into the skin for both local and systemic treatment. But concerns have been raised regarding their potential nanotoxicity. To understand the role of NLC composition in terms of cytotoxicity and pro-oxidant effects, we investigated cell viability and intracellular levels of ROS (reactive oxygen species) production in human dermal fibroblasts (HDF) incubated with five NLC formulations differing in their solid lipid composition. HDF and NLC were also exposed to UVA irradiation in order to evaluate the behavior of NLC under realistic environmental conditions which might promote their instability. Using the Guava via-count assay, all nanoparticles, except for those formulated with Compritol 888 ATO, showed a significant decrease in live cells and a parallel increase in apoptotic or dead cells compared to the control, either before and/or after UVA irradiation (18 J/cm(2)). NLC formulated with Geleol™ Mono Diglycerides resulted the most cytotoxic. A similar trend was also observed when intracellular ROS levels were measured in HDF incubated with NLC: there was increased ROS content compared to the control, further exacerbated following UVA. NLC formulated with Dynasan 118 were particularly susceptible to UVA exposure. The results indicate which could be the most suitable candidates for formulating NLC that are biocompatible and non-cytotoxic even when exposed to UVA and hence help direct future choices during the formulation strategies of these delivery systems. Of those tested, Compritol 888 ATO appears to be the best choice. PMID:26392245

  4. The effect of basic fibroblast growth factor on regeneration in a surgical wound model of rat submandibular glands.

    PubMed

    Kobayashi, Fumitaka; Matsuzaka, Kenichi; Inoue, Takashi

    2016-03-01

    This study developed an animal model of surgically wounded submandibular glands (SMGs) and investigated the effects of collagen gel with basic fibroblast growth factor (bFGF) on tissue regeneration of surgically wounded SMGs in vivo. The animal model was produced by creating a surgical wound using a 3-mm diameter biopsy punch in SMGs. The wound was filled with collagen gel with bFGF (bFGF group) or without bFGF (control group). In the animal model of surgically wounded SMGs, salivary glands without scar tissue around the wound area were observed with smaller areas of collagen gel. Small round and spindle-shape cells invaded the collagen gel in both groups after operation day (AOD) 5, and this invasion dramatically increased at AOD 7. Host tissue completely replaced the collagen gel at AOD 21. The invading immune cells in the group treated with collagen gel with bFGF were positive for vimentin, α-smooth muscle actin (αSMA), CD49f, c-kit and AQP5 at AOD 7. Similarly, the mRNA expression of vimentin, αSMA, CD49f, keratin19 and AQP5 was also increased. This study suggests that the use of collagen gels with bFGF improves salivary gland regeneration. PMID:27025261

  5. The effect of basic fibroblast growth factor on regeneration in a surgical wound model of rat submandibular glands

    PubMed Central

    Kobayashi, Fumitaka; Matsuzaka, Kenichi; Inoue, Takashi

    2016-01-01

    This study developed an animal model of surgically wounded submandibular glands (SMGs) and investigated the effects of collagen gel with basic fibroblast growth factor (bFGF) on tissue regeneration of surgically wounded SMGs in vivo. The animal model was produced by creating a surgical wound using a 3-mm diameter biopsy punch in SMGs. The wound was filled with collagen gel with bFGF (bFGF group) or without bFGF (control group). In the animal model of surgically wounded SMGs, salivary glands without scar tissue around the wound area were observed with smaller areas of collagen gel. Small round and spindle-shape cells invaded the collagen gel in both groups after operation day (AOD) 5, and this invasion dramatically increased at AOD 7. Host tissue completely replaced the collagen gel at AOD 21. The invading immune cells in the group treated with collagen gel with bFGF were positive for vimentin, α-smooth muscle actin (αSMA), CD49f, c-kit and AQP5 at AOD 7. Similarly, the mRNA expression of vimentin, αSMA, CD49f, keratin19 and AQP5 was also increased. This study suggests that the use of collagen gels with bFGF improves salivary gland regeneration. PMID:27025261

  6. Is there a Trojan-horse effect during magnetic nanoparticles and metalloid cocontamination of human dermal fibroblasts?

    PubMed

    Auffan, Melanie; Rose, Jerome; Proux, Olivier; Masion, Armand; Liu, Wei; Benameur, Laila; Ziarelli, Fabio; Botta, Alain; Chaneac, Corinne; Bottero, Jean-Yves

    2012-10-01

    This study investigates the issue of nanoparticles/pollutants cocontamination. By combining viability assays, physicochemical and structural analysis (to probe the As speciation and valence), we assessed how γFe(2)O(3) nanoparticles can affect the cytotoxicity, the intra- and extracellular speciation of As(III). Human dermal fibroblasts were contaminated with γFe(2)O(3) nanoparticles and As(III) considering two scenarios: (i) a simultaneous coinjection of the nanoparticles and As, and (ii) an injection of the nanoparticles after 24 h of As adsorption in water. In both scenarios, we did not notice significant changes on the nanoparticles surface charge (zeta potential ∼ -10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h. We demonstrated that the coinjection of γFe(2)O(3) nanoparticles and As in the cellular media strongly affects the complexation of the intracellular As with thiol groups. This significantly increases at low doses the cytotoxicity of the As nonadsorbed at the surface of the nanoparticles. However, once As is adsorbed at the surface the desorption is very weak in the culture medium. This fraction of As strongly adsorbed at the surface is significantly less cytotoxic than As itself. On the basis of our data and the thermodynamics, we demonstrated that any disturbance of the biotransformation mechanisms by the nanoparticles (i.e., surface complexation of thiol groups with the iron atoms) is likely to be responsible for the increase of the As adverse effects at low doses. PMID:22920588

  7. Contrasting effects of basic fibroblast growth factor and neurotrophin 3 on cell cycle kinetics of mouse cortical stem cells

    PubMed Central

    Lukaszewicz, Agnès; Savatier, Pierre; Cortay, Véronique; Kennedy, Henry; Dehay, Colette

    2002-01-01

    Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27kip1 and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27kip1 and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds. PMID:12151540

  8. Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

    PubMed Central

    Vahabi, Surena; Vaziri, Shahram; Torshabi, Maryam

    2015-01-01

    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. PMID:26877740

  9. The Nf1 Tumor Suppressor Regulates Mouse Skin Wound Healing, Fibroblast Proliferation, and Collagen Deposited by Fibroblasts

    PubMed Central

    Atit, Radhika P.; Crowe, Maria J.; Greenhalgh, David G.; Wenstrup, Richard J.; Ratner, Nancy

    2010-01-01

    Neurofibromatosis type 1 patients develop peripheral nerve tumors (neurofibromas) composed mainly of Schwann cells and fibroblasts, in an abundant collagen matrix produced by fibroblasts. Trauma has been proposed to trigger neurofibroma formation. To test if loss of the neurofibromatosis type 1 gene (Nf1) compromises fibroblast function in vivo following trauma, skin wounding was performed in Nf1 knockout mice. The pattern and amount of collagen-rich granulation bed tissue, manufactured by fibroblasts, was grossly abnormal in 60% of Nf1+/− wounds. Nf1 mutant fibroblasts showed cell autonomous abnormalities in collagen deposition in vitro that were not mimicked by Ras activation in fibroblasts, even though some Nf1 effects are mediated through Ras. Nf1+/− skin wound fibroblasts also proliferated past the normal wound maturation phase; this in vivo effect was potentiated by muscle injury. In vitro, Nf1+/− fibroblasts showed higher proliferation in 10% serum than Nf1+/+ fibroblasts. Macrophage-conditioned media or epidermal growth factor potentiated Nf1+/− fibroblast proliferation in vitro, demonstrating abnormal response of mutant fibroblasts to wound cytokines. Thus Nf1 is a key regulator of fibroblast responses to injury, and Nf1 mutation in mouse fibroblasts causes abnormalities characteristic of human neurofibromas. PMID:10383727

  10. Effect of meso-dihydroguaiaretic acid from Machilus thunbergii Sieb et Zucc on MMP-1 expression in heat shock-induced cultured primary human fibroblasts.

    PubMed

    Moon, Hyung-In; Moon, Hyungin; Jung, Jae-Chul

    2006-08-01

    Ethanol and aqueous extracts of Machilus thunbergii Sieb et Zucc (Lauraceae) used traditionally for the treatment of a variety of diseases were screened in vitro for MMP-1 inhibitory actions. Meso-dihydroguaiaretic acid (MDGA) from the stem bark of M. thunbergii showed a significant inhibition of matrix metalloproteinase (MMP)-1 in primary human fibroblasts by heat shock-induced. This study investigated the effect of MDGA isolated from M. thunbergii on heat shock-induced premature skin aging. MDGA reduced the expression of MMP-1 at the protein level in a dose-dependent manner in heat shock-induced cultured primary human fibroblasts. Taken together, these results show that MDGA can prevent the harmful effects of heat (and/or IR) that lead to skin aging. PMID:16775809

  11. IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts.

    PubMed

    Simmons, James G; Pucilowska, Jolanta B; Keku, Temitope O; Lund, P Kay

    2002-09-01

    Insulin-like growth factor I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-beta1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-beta1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of alpha-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-beta1 inhibited basal DNA synthesis. TGF-beta1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-beta1 but not IGF-I potently stimulated expression of alpha-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-beta1 attenuated stress fiber formation without reducing alpha-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-beta1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-beta1 pretreatment. IGF-I potently stimulated proliferation of TGF-beta1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-beta1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-beta1 and IGF-I, but they may act in concert to increase collagen deposition. PMID:12181198

  12. Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts.

    PubMed

    Claesson, Anna Kristina; Stenerlöw, Bo; Jacobsson, Lars; Elmroth, Kecke

    2007-03-01

    The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays. PMID:17316073

  13. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-Induced DNA Damages in Confluent Human Fibroblasts

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2016-01-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 µg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by foci and pattern counting of phosphorylated histone protein H2AX (?-H2AX). The cells on the ISS showed modestly increased average foci counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profile of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in ?-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect

  14. Characteristics of inositol trisphosphate-mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-11-05

    Ca/sup 2 +/ release triggered by inositol trisphosphate (Ins(1,4,5)P/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not greatly affected by the incubation temperature. The amount of Ca/sup 2 +/ released by Ins(1,4,5)P/sub 3/ was not affected by pH (6.5-8.0). La/sup 3 +/ (100 ..mu..M) markedly inhibited the effect of 1 ..mu..M Ins(1,4,5)P/sub 3/. The possibility that La/sup 3 +/ chelates Ins(1,4,5)P/sub 3/ cannot be excluded since the effect of La/sup 3 +/ could be overcome by increasing the Ins(1,4,5)P/sub 3/ concentration. Ins(1,4,5)P/sub 3/-mediated Ca/sup 2 +/ release showed a requirement for permeant cations in the incubation medium. Optimal release was observed with potassium gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibited Ca/sup 2 +/ release produced by Ins(1,4,5)P/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sup 2 -//sub 4/ were equally effective as inhibitors. Ins(1,4,5)P/sub 3/ also caused the release of /sup 54/Mn/sup 2 +/ and /sup 85/Sr/sup 2 +/ accumulated by the permeabilized hepatocytes. The results are consistent with Ins(1,4,5)P/sub 3/ promoting the membrane translocation of divalent cations through an ion channel rather than an ion carrier. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membranes. The transport systems responsible for these compensatory ion movements may represent a potential site for the regulation of the hormone-mediated Ca/sup 2 +/ signal.

  15. Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes.

    PubMed Central

    St-Denis, J F; Annabi, B; Khoury, H; van de Werve, G

    1995-01-01

    The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate. PMID:7646448

  16. Clinical implications of the growth-suppressive effects of chlorhexidine at low and high concentrations on human gingival fibroblasts and changes in morphology.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Kotwicka, Malgorzata; Urbaniak, Paulina; Nowak, Agnieszka; Skrzypczak-Jankun, Ewa; Jankun, Jerzy

    2016-06-01

    Chlorhexidine (CHX) is considered the gold standard in the antiseptic treatment of the oral cavity, due to its high antibactericidal capability. With the use of CHX mouth-rinse formulations, the bacteriostatic effects are maintained by the adsorption and prolonged release of CHX from oral surfaces. It was believed that antiplaque formation ability and the lack of systemic toxicity of CHX render it an excellent antiseptic in post-surgical dental treatment. However, recent studies have demonstrated that CHX exerts cytotoxic effects on human periodontal tissues, such as gingival fibroblasts and other cells. It also reduces gingival fibroblast adhesion to fibronectin and prevents fibroblast attachment to root surfaces, thus interfering with periodontal regeneration. In this study, using human gingival fibroblasts (HGFs), we investigated effects of CHX on the growth, morphology and proliferation of HGFs. We found that a low concentration (0.002%) of CHX does not interfere with the proliferation and morphology of HGFs. However, a higher concentration (≥0.04%) of CHX inhibits cell proliferation and to a certain extent, affects cell morphology in a time-dependent manner. A decrease in the percentage of cells in the G0/G1 phase and the accumulation of cells in the S phase following treatment with CHX also occurred in a dose-dependent manner. We thus concluded that CHX only at the concentration of 0.002% does not interfere with HGF growth, that is so critical to wound healing. Thus, the application of CHX in the post-surgical antiseptic treatment of the oral cavity should be limited. PMID:27082817

  17. Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts

    PubMed Central

    Han, Dong-Wook; Lee, Mi Hee; Kim, Bongju; Lee, Jun Jae; Hyon, Suong-Hyu; Park, Jong-Chul

    2012-01-01

    Considering the various pharmacological activities of epigallocatechin-3-O-gallate (EGCG) including anticancer, and anti-inflammatory, antidiabetic, and so forth, relatively less attention has been paid to the antiaging effect of EGCG on primary cells. In this study, the preventive effects of EGCG against serial passage-induced senescence were investigated in primary cells including rat vascular smooth muscle cells (RVSMCs), human dermal fibroblasts (HDFs), and human articular chondrocytes (HACs). The involvement of Sirt1 and acetylated p53 was examined as an underlying mechanism for the senescence preventive activity of EGCG in HDFs. All cells were employed with the initial passage number (PN) between 3 and 7. For inducing senescence, the cells were serially passaged at the predetermined times and intervals in the absence or presence of EGCG (50 or 100 μM). Serial passage-induced senescence in RVSMCs and HACs was able to be significantly prevented at 50 μM EGCG, while in HDFs, 100 μM EGCG could significantly prevent senescence and recover their cell cycle progression close to the normal level. Furthermore, EGCG was found to prevent serial passage- and H2O2-induced senescence in HDFs by suppressing p53 acetylation, but the Sirt1 activity was unaffected. In addition, proliferating HDFs showed similar cellular uptake of FITC-conjugated EGCG into the cytoplasm with their senescent counterparts but different nuclear translocation of it from them, which would partly account for the differential responses to EGCG in proliferating versus senescent cells. Taking these results into consideration, it is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent. PMID:23259030

  18. Effects of hydrogen peroxide on voltage-dependent K+ currents in human cardiac fibroblasts through protein kinase pathways

    PubMed Central

    Bae, Hyemi; Lee, Donghee; Kim, Young-Won; Choi, Jeongyoon; Lee, Hong Jun; Kim, Sang-Wook; Kim, Taeho; Noh, Yun-Hee; Ko, Jae-Hong; Bang, Hyoweon

    2016-01-01

    Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (KCa) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated KCa currents were blocked by iberiotoxin (IbTX, a large conductance KCa blocker). The H2O2-stimulating effect on large-conductance KCa (BKCa) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BKCa currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BKCa currents. Using RT-PCR and western blot analysis, three subtypes of KCa channels were detected in HCFs: BKCa channels, small-conductance KCa (SKCa) channels, and intermediate-conductance KCa (IKCa) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BKCa currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BKCa channels. PMID:27162486

  19. Effects of high density lipoprotein subfractions on cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells.

    PubMed

    Oram, J F

    1983-01-01

    Ultracentrifugally isolated high density lipoprotein (HDL) particles of d greater than 1.125 g/ml promote net transport of cholesterol from cultured cells. Consequently, when cultured human fibroblasts and arterial smooth muscle cells were incubated with HDL3 (d = 1.125-1.21 g/ml) and "very high" density lipoprotein (VHDL, d = 1.21-1.25 g/ml), low density lipoprotein (LDL) receptor activity was induced and the rate of LDL degradation by the cells was increased. Enhancement of LDL degradation by HDL3 and VHDL was sustained over incubation periods of 5 days at medium LDL concentrations greater than needed to saturate the LDL receptors. Even during these long-term incubations with LDL, HDL3 and VHDL caused marked reductions in cellular cholesterol content. Thus, an increase in the rate of cholesterol transport from cells may lead to a steady-state decrease in cellular cholesterol content and a sustained increase in the rate of clearance of LDL from the extracellular fluid. In contrast to the effects of HDL3 and VHDL, the major subclasses of HDL2 (HDL2b, d = 1.063-1.100 g/ml; HDL2a, d = 1.100-1.125 g/ml) did not promote net cholesterol transport from cells. Moreover, by apparent direct blockage of the effects that HDL3 and VHDL had on cholesterol transport, HDL2 reversed the increased rate of LDL degradation induced by HDL3 and VHDL. These results suggest that the relative proportion of HDL subfractions in the extracellular fluid may be an important determinant of both the rate of cholesterol transport from cells and the rate of receptor-mediated catabolism of LDL. PMID:6312947

  20. EFFECTS OF A CYTOKINETIC BLOCK ON THE DETERMINATION OF MICRONUCLEUS INDUCTION IN CHINESE HAMSTER LUNG FIBROBLASTS

    EPA Science Inventory

    Approximately 63,000 chemicals are found in our foods, drugs, consumer products and envrionment (11). here is proof that human exposure to some of these chemicals can have carcinogenic, teratogenic and mutagenic effects (11,5,14). overnment agencies and industry have developed a ...

  1. The effects of triiodothyronine, hydrocortisone and insulin on lipid synthesis by cultured fibroblasts preincubated in a serum-free medium.

    PubMed

    Amorosa, L F; Khachadurian, A K; Harris, J N; Schneider, S H; Fung, C H

    1984-02-01

    Studies of lipid metabolism in cell cultures are usually carried out after preincubation of cells in media containing lipoprotein-deficient or delipidated serum. The artifacts produced during delipidation prevent the standardization of assays and the study of the role of hormones on lipid metabolism. We studied the effects of triiodothyronine, hydrocortisone, insulin and their combination on cholesterol and fatty acid synthesis in cultured human skin fibroblasts preincubated for 24 h in an artificial medium (medium A) consisting of equal volumes of Dulbecco's modified Eagle's and Ham's F-12 media enriched with transferrin, biotin and calcium pantothenate. In cells preincubated in medium A the incorporation of acetate to cholesterol and the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase were much lower than in cells preincubated in standard medium containing lipoprotein-deficient serum. Addition of the three hormones caused a marked stimulation of the incorporation of acetate to cholesterol (from 3.1 to 17.7 pmol/min per mg protein), an activity similar to that in cells preincubated in lipoprotein-deficient serum plus hormones. The stimulatory effect of the hormones on HMG-CoA reductase activity was smaller, from 11 to 26 pmol/min per mg protein compared to 83 pmol/min per mg protein in cells preincubated in lipoprotein-deficient serum plus hormones. Most of the stimulatory effect was due to insulin. The lack of coordinate response between these two parameters in cells preincubated in artificial medium could not be explained by (a) stimulation of a post-mevalonate step as measured by the incorporation of mevalonate to cholesterol; (b) the in vitro inactivation of HMG-CoA reductase by phosphorylation: incubation of fibroblast microsomes with Escherichia coli alkaline phosphatase resulted in a decrease in HMG-CoA reductase activity, in contrast to an increase in hepatic microsomes; (c) the presence of inhibitors of HMG-CoA reductase in the microsomal

  2. HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

    PubMed Central

    Granato, M.; Zompetta, C.; Vescarelli, E.; Rizzello, C.; Cardi, A.; Valia, S.; Antonelli, G.; Marchese, C.; Torrisi, M. R.; Faggioni, A.; Cirone, M.

    2016-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis. PMID:27476557

  3. HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2.

    PubMed

    Granato, M; Zompetta, C; Vescarelli, E; Rizzello, C; Cardi, A; Valia, S; Antonelli, G; Marchese, C; Torrisi, M R; Faggioni, A; Cirone, M

    2016-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis. PMID:27476557

  4. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells.

    PubMed

    Salmenperä, Pertteli; Karhemo, Piia-Riitta; Räsänen, Kati; Laakkonen, Pirjo; Vaheri, Antti

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. PMID:27177832

  5. Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity.

    PubMed

    Tomek, Petr; Hrouzek, Pavel; Kuzma, Marek; Sýkora, Jan; Fiser, Radovan; Cerný, Jan; Novák, Petr; Bártová, Simona; Simek, Petr; Hof, Martin; Kavan, Daniel; Kopecký, Jirí

    2015-02-16

    There is mounting evidence that cyanobacterial lipopeptides can kill mammalian cells, presenting a hazard to human health. Unfortunately, their mechanism of toxicity is poorly understood. We have isolated new cyclic undecalipopeptides muscotoxin A and B containing unique lipophilicresidue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa). Muscotoxin B was not used for biological studies due to its poor yield. Muscotoxin A was cytotoxic to YAC-1, Sp/2, and HeLa cancer cell lines (LC(50) ranged from 9.9 to 13.2 μM after 24 h of exposure), causing membrane damage and influx of calcium ions. Subsequently, we studied this lytic mechanism using synthetic liposomes with encapsulated fluorescent probes. Muscotoxin A permeabilized liposomes composed exclusively of phospholipids, demonstrating that no proteins or carbohydrates present in biomembranes are essential for its activity. Paradoxically, the permeabilization activity of muscotoxin A was mediated by a significant reduction in membrane surface fluidity (stiffening), the opposite of that caused by synthetic detergents and cytolytic lipopeptide puwainaphycin F. At 25 °C, muscotoxin A disrupted liposomes with and without cholesterol/sphingomyelin; however, at 37 °C, it was selective against liposomes with cholesterol/sphingomyelin. It appears that both membrane fluidity and organization can affect the lytic activity of muscotoxin A. Our findings strengthen the evidence that cyanobacterial lipopeptides specifically disrupt mammalian cell membranes and bring new insights into the mechanism of this effect. PMID:25621379

  6. Protective effect of basic fibroblast growth factor on laser induced retinopathy

    PubMed Central

    Kartal, Unal; Koptagel, Emel; Bulut, H. Eray; Erdogan, Haydar

    2013-01-01

    AIM To investigate the side effects of the commonly used laser treatment along with testing the neuroprotective effect of bFGF on a potential retinal impairment. METHODS To do this, 30 chinchilla pigmented adult male rabbits were divided into the control and experimental groups. The control and experimental groups underwent both laser application and bFGF treatment. The retinal tissue impairment and its renewal rate were tested under the light and electron microscopical levels. RESULTS The focal laser application on rabbit eyes caused morphological alterations both in the application region and in the neighbouring areas. In the damaged areas, the outer nuclear layer of the neural retina was almost disappeared, retina pigment epithelium was interrupted, the retina pigment epithelium migrated intraretinally, and the damaged region along with neighbouring areas seemed to be not separated. bFGF application just after the laser photocoagulation, revealed better results in application areas. CONCLUSION It could be suggested that the bFGF application following laser photocoagulation might have protective, repairing and wound healing effects on the retina. PMID:24392319

  7. Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

    SciTech Connect

    Nakayama, Fumiaki; Umeda, Sachiko; Yasuda, Takeshi; Asada, Masahiro; Motomura, Kaori; Suzuki, Masashi; Zakrzewska, Malgorzata; Imamura, Toru; Imai, Takashi

    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

  8. Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Honda, Ryosuke; Hokari, Yutaro; Takashima, Keisuke; Kanzaki, Makoto; Kaneko, Toshiro

    2016-08-01

    Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor(s), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OHaq), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (<1 mm), and the plasma-produced OHaq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OHaq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OHaq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool.

  9. Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier.

    PubMed

    Bonakdar, Mohammad; Wasson, Elisa M; Lee, Yong W; Davalos, Rafael V

    2016-01-19

    The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway. PMID:26789772

  10. Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2.

    PubMed

    Villalpando Rodriguez, Gloria E; Torriglia, Alicia

    2013-10-01

    In light induced retinal degeneration (LIRD) photoreceptor cell death is mediated by caspase independent mechanisms. The activation of LEI/L-DNase II pathway in this model, is due to cathepsin D release from lysosomes, although the underlying mechanism remains poorly understood. In this paper we studied the involvement of calpains in lysosomal permeabilization. We investigated, for the first time, the calpain targets at lysosomal membrane level. We found that calpain 1 is responsible for lysosomal permeabilization by cleavage of the lysosomal associated membrane protein 2 (LAMP 2). Moreover, LAMP 2 degradation and lysosomal permeabilization were rescued by calpain inhibition and the use of MEF(-/-)lamp 2 cells indicates that the cleavage of LAMP 2A is essential for this permeabilization. Finally, we found that LAMP 2 is cleaved in LIRD, suggesting that the mechanism of calpain induced lysosomal permeabilization is not exclusive of a single cell death model. Overall, these data shed new light on understanding the mechanisms of lysosomal and caspase-independent cell death and point to the original targets for development of the new therapeutic protocols. PMID:23747342

  11. Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation.

    PubMed

    Kohli, Vikram; Acker, Jason P; Elezzabi, Abdulhakem Y

    2005-12-30

    Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein we report on the uniqueness of using high-intensity femtosecond laser pulses for reversibly permeabilizing mammalian cells for biopreservation applications. When mammalian cells were suspended in a impermeable hyperosmotic cryoprotectant sucrose solution, femtosecond laser pulses were used to transiently permeabilize cells for cytoplasmic solute uptake. The kinetics of cells exposed to 0.2, 0.3, 0.4, and 0.5 M sucrose, following permeabilization, were measured using video microscopy, and post-permeabilization survival was determined by a dual fluorescence membrane integrity assay. Using appropriate laser parameters, we observed the highest cell survival for 0.2 M sucrose solution (>90%), with a progressive decline in cell survival towards higher concentrations. Using diffusion equations describing the transport of solutes, the intracellular osmolarity at the inner surface of the membrane (x = 10 nm) and to a diffusive length of x = 10 microm was estimated, and a high loading efficiency (>98% for x = 10 nm and >70% for x = 10 microm) was calculated for cells suspended in 0.2 M sucrose. This is the first report of using femtosecond laser pulses for permeabilizing cells in the presence of cryoprotectants for biopreservation applications. PMID:16189821

  12. High production in E. coli of biologically active recombinant human fibroblast growth factor 20 and its neuroprotective effects.

    PubMed

    Tian, Haishan; Zhao, Yang; Chen, Nazi; Wu, Meiyu; Gong, Weiyue; Zheng, Jie; Fernig, David G; Jungbauer, Alois; Wang, Dezhong; Li, Xiaokun; Jiang, Chao

    2016-04-01

    Fibroblast growth factor 20 (FGF20) has a wide range of biological activities; its expression is most pronounced in neural tissues where it has functions in development and neuroprotection. Given these activities, interest in the clinical applications of FGF20 is rising, which will lead to increasing demand for active recombinant human FGF20 (rhFGF20). To improve the production of rhFGF20, an artificial gene encoding fgf20 was cloned into pET3a and expressed in E. coli BL21(DE3)pLysS. By optimizing induction conditions, we successfully induced large amounts of insoluble rhFGF20. Following solubilization and refolding of the rhFGF20 from inclusion bodies, it was purified by HiTrap heparin affinity chromatography to a purity of over 96 % with a yield of 218 mg rhFGF20/100 g wet cells. The purified rhFGF20 could stimulate proliferation of both NIH 3T3 cells and PC-12 cells, measured by the MTT assay. In a model of Aβ25-35-induced apoptosis on PC-12 cells, rhFGF20 had a clear protective effect. RT-PCR and Western blot analysis of apoptosis-related genes and proteins revealed that the FGF20-derived protective mechanism was likely due to the relief of endoplasmic reticulum stress (ER stress). In conclusion, the approach described here may be a better means to produce active rhFGF20 in good quantity, thereby allowing for its future pharmacological and clinical use. PMID:26603761

  13. Effect of Control-released Basic Fibroblast Growth Factor Incorporated in β-Tricalcium Phosphate for Murine Cranial Model

    PubMed Central

    Shimizu, Azusa; Tajima, Satoshi; Tobita, Morikuni; Tanaka, Rica; Tabata, Yasuhiko

    2014-01-01

    Background: β-Tricalcium phosphate (β-TCP) is used clinically as a bone substitute, but complete osteoinduction is slow. Basic fibroblast growth factor (bFGF) is important in bone regeneration, but the biological effects are very limited because of the short half-life of the free form. Incorporation in gelatin allows slow release of growth factors during degradation. The present study evaluated whether control-released bFGF incorporated in β-TCP can promote bone regeneration in a murine cranial defect model. Methods: Bilateral cranial defects of 4 mm in diameter were made in 10-week-old male Sprague-Dawley rats treated as follows: group 1, 20 μl saline as control; group 2, β-TCP disk in 20 μl saline; group 3, β-TCP disk in 50 μg bFGF solution; and group 4, β-TCP disk in 50 μg bFGF-containing gelatin hydrogel (n = 6 each). Histological and imaging analyses were performed at 1, 2, and 4 weeks after surgery. Results: The computed tomography value was lower in groups 3 and 4, whereas the rate of osteogenesis was higher histologically in group 4 than in the other groups. The appearance of tartrate-resistant acid phosphate–positive cells and osteocalcin-positive cells and disappearance of osteopontin-positive cells occurred earlier in group 4 than in the other groups. Conclusions: These findings suggest that control-released bFGF incorporated in β-TCP can accelerate bone regeneration in the murine cranial defect model and may be promising for the clinical treatment of cranial defects. PMID:25289319

  14. Effects of flavonoids on expression of genes involved in cell cycle regulation and DNA replication in human fibroblasts.

    PubMed

    Moskot, Marta; Jakóbkiewicz-Banecka, Joanna; Smolińska, Elwira; Piotrowska, Ewa; Węgrzyn, Grzegorz; Gabig-Cimińska, Magdalena

    2015-09-01

    Flavonoids have been studied as potential agents in medicine for many years. Among them, genistein was found to be active in various biological systems, mainly in prevention of cancer. Our recent work supported the idea that genistein also impacts multiple cellular processes in healthy fibroblasts; however, its effects on cell cycle-related pathways remained to be elucidated. Thus, in this work, high throughput screening with microarrays coupled to real-time quantitative Reverse Transcription PCR analyses was employed to study the changes in expression of key genes associated with cell cycle regulation and/or DNA replication in response to genistein, kaempferol, daidzein, and mixtures of genistein and either kaempferol or daidzein. Among them, genistein was found as the most significantly modulating, in a time- and dose-dependent manner, compound of activity of studied genes, whose products are involved in different phases of the cell cycle and/or in regulatory processes important for DNA replication and cell growth. It considerably reduced the efficiency of expression of genes coding for MCM2-7 and MCM10 helicases, as well as some other proteins involved in the S phase control. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and GADD45A genes, as well as down-regulation of several mRNAs specific for this stage, demonstrated by transcriptomic assessments. We believe that studies described in this paper will be helpful in elucidating molecular mechanisms of action of genistein as modulator of cell cycle and inhibitor of DNA replication in humans. PMID:26003441

  15. Protective effect of basic fibroblast growth factor on retinal injury induced by argon laser photocoagulation

    NASA Astrophysics Data System (ADS)

    Chen, P.; Zhang, C. P.; San, Q.; Wang, C. Z.; Yang, Z. F.; Kang, H. X.; Qian, H. W.

    2010-12-01

    Laser photocoagulation treatment is often complicated by a side effect of visual impairment, which is caused by the unavoidable laser-induced retinal destruction. At present no specific is found to cure this retinopathy. The aim of this study was to observe the neuroprotective effect of bFGF on laser-induced retinal injury. Chinchilla rabbits were divided into three groups and argon laser lesions were created in the retinas. Then bFGF or dexamethasone, a widely used ophthalmic preparation, or saline was given severally by retrobulbar injection. The retinal lesions were evaluated histologically and morphometrically, and visual function was examined by ERG. The results showed that bFGF administration better preserved morphology of retinal photoreceptors and significantly diminished the area of the lesions. Furthermore, bFGF promoted the restoration of the ERG b-wave amplitude. In rabbits treated with dexamethasone, however, the lesions showed almost no ameliorative changes. This is the first study to investigate the potential role of bFGF as a remedial agent in laser photocoagulation treatment. These findings suggest that bFGF has significant neuroprotective properties in the retina and this type of neuroprotection may be of clinical significance in reducing iatrogenic laser-induced retinal injuries in humans.

  16. Protective effects of adipose-derived stem cells secretome on human dermal fibroblasts from ageing damages

    PubMed Central

    Wang, Ting; Guo, Shu; Liu, Xuehui; Xv, Nan; Zhang, Shuangyi

    2015-01-01

    Background: Combined effects of intrinsic and extrinsic ageing factors on skin tissue and the therapies have been rarely studied before. ADSCs have gained popularity in anti-ageing field, which may provide promising methods to fight against skin ageing. Objective: To find out the fate of HDFs exposed to intrinsic or extrinsic ageing factors or both of them and further examine the impacts of ADSC-CM on the damaged HDFs. Methods: We irradiated HDFs with UVB at different senescent levels, and then treated them with ADSC-CM. After 48 h, we detected cellular proliferative activity, morphology, SA-β-Gal expression, apoptosis, mRNA expression of collagen I, collagen III and elastin. Results: Intrinsic ageing factors inhibited cellular proliferation, increased senescent ratio and reduced mRNA expression of collagen I, collagen III and elastin, so did UVB, except for its induction of elastin mRNA expression. Furthermore, ADSC-CM treatment can slightly or significantly improve cellular proliferative activity and restore functions both in irradiated and non-irradiated HDFs. Besides, ADSC-CM treatment decreased cellular apoptosis and senescence induced by UVB but had no obvious effect on cellular senescence induced by intrinsic ageing factors. The results were similar in three generations of HDFs, yet in different degrees. Conclusions: The results suggest that ADSCs secretome protect HDFs from ageing damages but with some limitations. PMID:26884843

  17. Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

    PubMed

    Stevens, Roy H; de Moura Martins Lobo Dos Santos, Caroline; Zuanazzi, David; de Accioly Mattos, Marcelo Barbosas; Ferreira, Davis Fernandes; Kachlany, Scott C; Tinoco, Eduardo M B

    2013-01-01

    Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens. PMID:23022667

  18. Effects of a vegetable extract from Lupinus albus (LU105) on the production of matrix metalloproteinases (MMP1, MMP2, MMP9) and tissue inhibitor of metalloproteinases (TIMP1, TIMP2) by human gingival fibroblasts in culture.

    PubMed

    Gaultier, F; Foucault-Bertaud, A; Lamy, E; Ejeil, A L; Dridi, S M; Piccardi, N; Piccirilli, A; Msika, P; Godeau, G; Gogly, B

    2003-12-01

    This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva. PMID:12802622

  19. Photodynamic effects of zinc oxide nanowires in skin cancer and fibroblast.

    PubMed

    Fakhar-e-Alam, Muhammad; Kishwar, S; Willander, M

    2014-05-01

    Cytotoxic effects of zinc oxide (ZnO) nanomaterials, individual and conjugated with a photosensitizer (protoporphyrin IX), were studied in the presence and absence of ultraviolet light exposure (240 nm of light wavelength for a very short time exposure) in cell cultures of human normal and cancerous skin models. Zinc Oxide nanowires (ZnO NWs) were grown on the capillary tip and conjugated with protoporphyrin IX (PpIX). This coated tip was used as tool/pointer for intracellular drug delivery protocol in suggested normal as well as carcinogenic cellular models. After true delivery of optimal drug, the labelled biological model was irradiated with UV-A, which led to a loss of mitochondrial membrane potential, as tested by neutral red assay (NRA). PMID:24338134

  20. 1,3-dinitrobenzene: toxic effects in vivo and in vitro. [Rats; Fibroblasts

    SciTech Connect

    Cody, T.E.; Witherup, S.; Hastings, L.; Stemmer, K.; Christian, R.T.

    1981-05-01

    When 1,3-dinitrobenzene (1,3-DNB) was given to rats orally as a 1% suspension in corn oil, the LD50 was 83 mg/kg with fiducial limits 56 to 124 mg/kg. The compound was equally toxic in both sexes. Signs of toxicity included reduction in ambulatory motion, ataxia, weaknes, dyspnea, rapid heartbeat, cyanosis, coma, and eventual respiratory failure. When 1,3-DNB was added to the daily drinking water in concentrations of 50, 100 and 200 mg/l for 8 wk, growth rate was reduced in both sexes and at 200 mg/l 1,3-DNB in the water supply the animals lost weight. There were mild, consistent reductions in hematocrit and hemoglobin values. Enlarged spleens were present in both sexes at all concentrations. Testicular atrophy was evident but there was no effect on the ovaries. When 3, 8, or 20 mg/l 1,3-DNB was provided daily in drinking water for 16 wk, no signs of acute toxicity were seen. Growth of females receiving 20 mg/l was retarded progressively after 8 wk of exposure; growth was normal in the other groups. Behavioral abnormalities, in the form of increased activity in running wheels, were noted in male rats exposed to 3 and 8 mg/l 1,3-DNB in drinking water for 90 d. Minimal effects on cells in vitro were noted at 3-8 mg 1,3-DNB per liter of medium. Growth reduction by 50% was observed at 20 to 25 mg/l. All cells were killed when the concentration of 1,3-DNB to which they were exposed exceeded 56 mg/l.

  1. Effects of cytokines and periodontopathic bacteria on the leukocyte function-associated antigen 1/intercellular adhesion molecule 1 pathway in gingival fibroblasts in adult periodontitis.

    PubMed

    Hayashi, J; Saito, I; Ishikawa, I; Miyasaka, N

    1994-12-01

    We investigated the effects of inflammatory cytokines and periodontopathic bacteria on expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1, and E-selectin (endothelial leukocyte adhesion molecule 1) in cultured human gingival fibroblasts (HGF). Cell surface ICAM-1 was upregulated on HGF under transcriptional control by exposure not only to interleukin-1 beta, tumor necrosis factor alpha, and gamma interferon but also to sonic extracts prepared from Porphyromonas gingivalis and Prevotella intermedia (nigrescens) and lipopolysaccharides from Escherichia coli. However, these stimuli induced only minimal expression of vascular cell adhesion molecule 1 and E-selectin on HGF. Binding assays using HGF and Molt 4, the human T-cell leukemia cell line, showed induced ICAM-1 to be functional, and the increased binding was blocked by a combination of monoclonal antibodies against ICAM-1 and leukocyte function-associated antigen 1. Furthermore, gingival tissues from adult periodontitis patients showed increased mRNA expression of ICAM-1 compared with that in tissues from normal healthy donors. In immunohistological analysis, we also observed in vivo that the expression of ICAM-1 on fibroblasts in adult periodontitis tissues was greater than that in normal gingiva. Thus, the overexpression of ICAM-1 on gingival fibroblasts induced by cytokines and periodontopathic bacteria is speculated to be deeply involved in the accumulation and retention of leukocyte function-associated antigen 1-bearing leukocytes in adult periodontitis lesions. PMID:7525481

  2. Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

    PubMed Central

    Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

    2016-01-01

    Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540

  3. Cellular Internalization of Fibroblast Growth Factor-12 Exerts Radioprotective Effects on Intestinal Radiation Damage Independently of FGFR Signaling

    SciTech Connect

    Nakayama, Fumiaki; Umeda, Sachiko; Yasuda, Takeshi; Fujita, Mayumi; Asada, Masahiro; Meineke, Viktor; Imamura, Toru; Imai, Takashi

    2014-02-01

    Purpose: Several fibroblast growth factors (FGFs) were shown to inhibit radiation-induced tissue damage through FGF receptor (FGFR) signaling; however, this signaling was also found to be involved in the pathogenesis of several malignant tumors. In contrast, FGF12 cannot activate any FGFRs. Instead, FGF12 can be internalized readily into cells using 2 cell-penetrating peptide domains (CPP-M, CPP-C). Therefore, this study focused on clarifying the role of FGF12 internalization in protection against radiation-induced intestinal injury. Methods and Materials: Each FGF or peptide was administered intraperitoneally to BALB/c mice in the absence of heparin 24 hours before or after total body irradiation with γ rays at 9 to 12 Gy. Several radioprotective effects were examined in the jejunum. Results: Administration of FGF12 after radiation exposure was as effective as pretreatment in significantly promoting intestinal regeneration, proliferation of crypt cells, and epithelial differentiation. Two domains, comprising amino acid residues 80 to 109 and 140 to 169 of FGF12B, were identified as being responsible for the radioprotective activity, so that deletion of both domains from FGF12B resulted in a reduction in activity. Interestingly, these regions included the CPP-M and CPP-C domains, respectively; however, CPP-C by itself did not show an antiapoptotic effect. In addition, FGF1, prototypic FGF, possesses a domain corresponding to CPP-M, whereas it lacks CPP-C, so the fusion of FGF1 with CPP-C (FGF1/CPP-C) enhanced cellular internalization and increased radioprotective activity. However, FGF1/CPP-C reduced in vitro mitogenic activity through FGFRs compared with FGF1, implying that FGFR signaling might not be essential for promoting the radioprotective effect of FGF1/CPP-C. In addition, internalized FGF12 suppressed the activation of p38α after irradiation, resulting in reduced radiation-induced apoptosis. Conclusions: These findings indicate that FGF12 can protect the

  4. Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function.

    PubMed

    van Nieuwenhoven, Frans A; Hemmings, Karen E; Porter, Karen E; Turner, Neil A

    2013-01-01

    During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6-24h by real-time RT-PCR, while protein levels were analyzed at 24-48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1. PMID:23583823

  5. Permeabilization of yeast Saccharomyces cerevisiae cell walls using nanosecond high power electrical pulses

    NASA Astrophysics Data System (ADS)

    Stirke, A.; Zimkus, A.; Balevicius, S.; Stankevic, V.; Ramanaviciene, A.; Ramanavicius, A.; Zurauskiene, N.

    2014-12-01

    The electrical field-induced changes of the yeast Saccharomyces cerevisiae cells permeabilization to tetraphenylphosphonium (TPP+) ions were studied using square-shaped, nanosecond duration high power electrical pulses. It was obtained that pulses having durations ranging from 10 ns to 60 ns, and generating electric field strengths up to 190 kV/cm significantly (up to 65 times) increase the absorption rate of TPP+ ions without any detectible influence on the yeast cell viability. The modelling of the TPP+ absorption process using a second order rate equation demonstrates that depending on the duration of the pulses, yeast cell clusters of different sizes are homogeniously permeabilized. It was concluded, that nanosecond pulse-induced permeabilization can be applied to increase the operational speed of whole cell biosensors.

  6. Fractal morphology of Beta vulgaris L. cell suspension culture permeabilized with Triton X-100®

    NASA Astrophysics Data System (ADS)

    Arenas-Ocampo, M.; Alamilla-Beltrán, L.; Vanegas-Espinoza, P. E.; Camacho-Díaz, B. H.; Campos-Mendiola, R.; Gutiérrez-López, G.; Jiménez-Aparicio, A.

    2012-02-01

    In this work, morphology of Beta vulgaris L. cells permeabilized with 0.7mM of Triton X-100® was evaluated using digital image processing and concepts of fractal dimension (perimeter- area relations). Important morphometric changes were found when the contact-time with chemical agent was increased. The size of cells decreased, the cells lost the roundness and their shape was more sinuous; this behaviour was a result of a probable shrinkage caused by the excess of exposure with the permeabilization agent. Morphology of B. vulgaris cells after permeabilization, exhibited a fractal nature since the slope of the ratio of the logarithm of the perimeter vs logarithm of the area was higher than unit. Fractal geometry of the cell morphology was affected as a result of the exposure to Triton X-100®. Those changes can be attributed to the loss of turgor and structure of the cell wall.

  7. Elevated fibroblast growth factor 23 exerts its effects on placenta and regulates vitamin D metabolism in pregnancy of Hyp mice.

    PubMed

    Ohata, Yasuhisa; Yamazaki, Miwa; Kawai, Masanobu; Tsugawa, Naoko; Tachikawa, Kanako; Koinuma, Tomoko; Miyagawa, Kazuaki; Kimoto, Akihito; Nakayama, Masahiro; Namba, Noriyuki; Yamamoto, Hironori; Okano, Toshio; Ozono, Keiichi; Michigami, Toshimi

    2014-07-01

    Fibroblast growth factor 23 (FGF23) functions in an endocrine fashion and requires α-Klotho to exert its effects on the target organs. We have recently demonstrated that the human placenta also expresses α-Klotho, which led us to hypothesize that FGF23 may exert effects on the placenta. Immunohistochemical analysis demonstrated the expression of FGF receptor 1 (FGFR1) as well as that of α-Klotho in the feto-maternal interface of both mouse and human normal-term placentas, which suggested that these areas might be receptive to FGF23. Therefore, we next investigated whether FGF23 has some roles in the placenta using Hyp mice with high levels of circulating FGF23. Hyp and wild-type (WT) females were mated with WT males, and the mothers and their male fetuses were analyzed. FGF23 levels in Hyp mothers were elevated. FGF23 levels were about 20-fold higher in Hyp fetuses than in Hyp mothers, whereas WT fetuses from Hyp mothers exhibited low levels of FGF23, as did fetuses from WT mothers. We analyzed the placental gene expression and found that the expression of Cyp24a1 encoding 25OHD-24-hydroxylase, a target gene for FGF23 in the kidney, was increased in the placentas of fetuses from Hyp mothers compared with fetuses from WT mothers. In an organ culture of WT placentas, treatment with plasma from Hyp mothers markedly increased the expression of Cyp24a1, which was abolished by the simultaneous addition of anti-FGF23 neutralizing antibody. The direct injection of recombinant FGF23 into WT placentas induced the expression of Cyp24a1. The increase in the placental expression of Cyp24a1 in fetuses from Hyp mothers resulted in decreased plasma 25-hydroxyvitamin D levels. These results suggest that increased levels of circulating FGF23 in pathological conditions such as Hyp mice exerts direct effects on the placenta and affects fetal vitamin D metabolism via the regulation of Cyp24a1 expression. PMID:24470103

  8. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

    2016-07-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

  9. All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713.

    PubMed

    Patel, Hiren; Tscheka, Clemens; Edwards, Katarina; Karlsson, Göran; Heerklotz, Heiko

    2011-08-01

    The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance. PMID:21545788

  10. (-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization

    PubMed Central

    LeGendre, Onica; Breslin, Paul AS; Foster, David A

    2015-01-01

    (-)-Oleocanthal (OC), a phenolic compound present in extra-virgin olive oil (EVOO), has been implicated in the health benefits associated with diets rich in EVOO. We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in the absence of serum. OC treatment of non-transformed cells suppressed their proliferation but did not cause cell death. OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP). We provide evidence that OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins required for lysosomal membrane stability. The data presented here indicate that cancer cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells, are susceptible to cell death induced by lysosomotropic agents. Therefore, targeting lysosomal membrane stability represents a novel approach for the induction of cancer-specific cell death. PMID:26380379

  11. HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

    PubMed Central

    Lecoeur, H; Borgne-Sanchez, A; Chaloin, O; El-Khoury, R; Brabant, M; Langonné, A; Porceddu, M; Brière, J-J; Buron, N; Rebouillat, D; Péchoux, C; Deniaud, A; Brenner, C; Briand, J-P; Muller, S; Rustin, P; Jacotot, E

    2012-01-01

    The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor. PMID:22419111

  12. Cytotoxicity of silver dressings on diabetic fibroblasts.

    PubMed

    Zou, Shi-Bo; Yoon, Won-Young; Han, Seung-Kyu; Jeong, Seong-Ho; Cui, Zheng-Jun; Kim, Woo-Kyung

    2013-06-01

    A large number of silver-based dressings are commonly used in the management of chronic wounds that are at risk of infection, including diabetic foot ulcers. However, there are still controversies regarding the toxicity of silver dressings on wound healing. The purpose of this study was to objectively test the cytotoxicity of silver dressings on human diabetic fibroblasts. Human diabetic fibroblasts were obtained from the foot skin of four diabetic foot ulcer patients and cultured. The effect of five silver-containing dressing products (Aquacel Ag, Acticoat*Absorbent, Medifoam Ag, Biatain Ag and PolyMem Ag) and their comparable silver-free dressing products on morphology, proliferation and collagen synthesis of the cultured human diabetic fibroblasts were compared in vitro. In addition, extracts of each dressing were tested in order to examine the effect of other chemical components found in the dressings on cytotoxicity. The diabetic fibroblasts cultured with each silver-free dressing adopted the typical dendritic and fusiform shape. On the other hand, the diabetic fibroblasts did not adopt this typical morphology when treated with the different silver dressings. All silver dressings tested in the study reduced the viability of the diabetic fibroblasts and collagen synthesis by 54-70 and 48-68%, respectively, when compared to silver-free dressings. Silver dressings significantly changed the cell morphology and decreased cell proliferation and collagen synthesis of diabetic fibroblasts. Therefore, silver dressings should be used with caution when treating diabetic wounds. PMID:22533495

  13. Turbulent states and their transitions in mathematical models for ventricular tissue: The effects of random interstitial fibroblasts

    NASA Astrophysics Data System (ADS)

    Nayak, Alok Ranjan; Pandit, Rahul

    2015-09-01

    We study the dynamical behaviors of two types of spiral- and scroll-wave turbulence states, respectively, in two-dimensional (2D) and three-dimensional (3D) mathematical models, of human, ventricular, myocyte cells that are attached to randomly distributed interstitial fibroblasts; these turbulence states are promoted by (a) the steep slope of the action-potential-duration-restitution (APDR) plot or (b) early afterdepolarizations (EADs). Our single-cell study shows that (1) the myocyte-fibroblast (MF) coupling Gj and (2) the number Nf of fibroblasts in an MF unit lower the steepness of the APDR slope and eliminate the EAD behaviors of myocytes; we explore the pacing dependence of such EAD suppression. In our 2D simulations, we observe that a spiral-turbulence (ST) state evolves into a state with a single, rotating spiral (RS) if either (a) Gj is large or (b) the maximum possible number of fibroblasts per myocyte Nfmax is large. We also observe that the minimum value of Gj, for the transition from the ST to the RS state, decreases as Nfmax increases. We find that, for the steep-APDR-induced ST state, once the MF coupling suppresses ST, the rotation period of a spiral in the RS state increases as (1) Gj increases, with fixed Nfmax, and (2) Nfmax increases, with fixed Gj. We obtain the boundary between ST and RS stability regions in the Nfmax-Gj plane. In particular, for low values of Nfmax, the value of Gj, at the ST-RS boundary, depends on the realization of the randomly distributed fibroblasts; this dependence decreases as Nfmax increases. Our 3D studies show a similar transition from scroll-wave turbulence to a single, rotating, scroll-wave state because of the MF coupling. We examine the experimental implications of our study and propose that the suppression (a) of the steep slope of the APDR or (b) EADs can eliminate spiral- and scroll-wave turbulence in heterogeneous cardiac tissue, which has randomly distributed fibroblasts.

  14. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    PubMed Central

    Talaei-Khozani, Tahereh; Heidari, Fatemeh; Esmaeilpour, Tahereh; Vojdani, Zahra; Mostafavi-Pour, Zohrah; Rohani, Leili

    2014-01-01

    Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function. PMID:24753644

  15. Effect of Proline-Containing Oligopeptides PGP and RGP on Proliferative and Protein-Synthesizing Activity of Cultured Pulmonary Fibroblasts under Conditions of Oxidative Stress.

    PubMed

    Tolstenok, I V; Fleishman, M Yu; Sazonova, E N; Lebed'ko, O A; Maltseva, I M; Myasoedov, N F; Timoshin, S S

    2016-05-01

    We studied the effect of glyprolines Pro-Gly-Pro (PGP) and Arg-Gly-Pro (RGP) on the primary culture of pulmonary fibroblasts from newborn albino rats under normal conditions and during oxidative stress. Under physiological conditions, the peptides had no effect on parameters of cell division. Hydrogen peroxide induced intensive oxidative stress accompanied by suppression of protein-synthesizing function. When hydrogen peroxide was added to the culture containing the test peptides, correction of the oxidative status was observed accompanied by activation of DNA-synthesizing activity and inhibition of lucigenin-dependent chemiluminescence. PMID:27265140

  16. Guanine nucleotide is essential and Ca2+ is a modulator in the exocytotic reaction of permeabilized rat mast cells.

    PubMed Central

    Lillie, T H; Gomperts, B D

    1992-01-01

    Exocytosis from metabolically depleted permeabilized rat mast cells was measured in response to provision of Ca2+ and guanine nucleotide [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])]. For cells permeabilized in simple salt solutions (NaCl), both of these effectors were required to induce secretion. Exclusion of Mg2+ caused an increase in both the sensitivity of the system to GTP and the extent of secretion elicited, while having no such effects on secretion induced by GTP[S]. The effect of Mg2+ depletion on the ability of GTP to stimulate secretion is probably due to the dependence on Mg2+ of the GTPase activity of GE (a postulated GTP-binding protein which mediates exocytosis). This argues that a persistent stimulus to the G-protein is required to support secretion. Affinity for both GTP[S] and GTP is enhanced when the cells are permeabilized in zwitterionic electrolytes (glutamate, gamma-aminobutyric acid, glycine) instead of NaCl. Under these conditions, secretion occurs in response to provision of either GTP[S] [in the effective absence of Ca2+ (pCa 9)] or Ca2+ (in the absence of guanine nucleotide). Secretion induced by GTP[S] is strongly promoted by the presence of Mg2+ at concentrations in the millimolar range; this promotion by Mg2+ declines as the concentration of Ca2+ is elevated towards pCa 7. At pCa 6, Mg2+ is without effect. Ca(2+)-induced secretion requires the provision of MgATP. Since this is further enhanced by low concentrations (< 100 microM) and then inhibited by high concentrations of GDP, the essential role of ATP is likely to be in the maintenance of GTP via transphosphorylation by a nucleoside diphosphate kinase reaction. Thus, under conditions of high affinity (glutamate environment), GTP[S] alone is capable of inducing exocytosis. Ca2+ acts in concert with guanine nucleotides: it enhances the rate and extent of secretion and increases the affinity for Mg2+ and guanine nucleotides in the activation of the GTP-binding protein (GE

  17. Transformable Peptide Nanocarriers for Expeditious Drug Release and Effective Cancer Therapy via Cancer-Associated Fibroblast Activation.

    PubMed

    Ji, Tianjiao; Zhao, Ying; Ding, Yanping; Wang, Jing; Zhao, Ruifang; Lang, Jiayan; Qin, Hao; Liu, Xiaoman; Shi, Jian; Tao, Ning; Qin, Zhihai; Nie, Guangjun; Zhao, Yuliang

    2016-01-18

    A novel cleavable amphiphilic peptide (CAP) was designed to be specifically responsive to fibroblast activation protein-α (FAP-α), a protease specifically expressed on the surface of cancer-associated fibroblasts. The CAP self-assembled into fiber-like nanostructures in solution, while the presence of hydrophobic chemotherapeutic drugs readily transformed the assemblies into drug-loaded spherical nanoparticles. The disassembly of these nanoparticles (CAP-NPs) upon FAP-α cleavage resulted in rapid and efficient release of the encapsulated drugs specifically at tumor sites. This Transformers-like drug delivery strategy could allow them to disrupt the stromal barrier and enhance local drug accumulation. Therapeutic results suggested that drug-loaded CAP-NPs hold promising tumor specificity and therapeutic efficacy for various solid tumor models, confirming its potential utility and versatility in antitumor therapy. PMID:26283097

  18. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  19. The Effects of Fibroblast Co-Culture and Activin A on in vitro Growth of Mouse Preantral Follicles

    PubMed Central

    Karimpour Malekshah, Abbasali; Heidari, Mahmoud; Parivar, Kazem; Azami, Nasrin Sadat

    2014-01-01

    Background: This study was conducted to evaluate fibroblast co-culture and Activin A on in vitro maturation and fertilization of mouse preantral follicles. Methods: The ovaries from 12-14-day-old mice were dissected, and 120-150 μm preantral follicles were cultured individually in α-MEM as based medium for 12 days. A total number of 456 follicles were cultured in four conditions: (i) base medium as control group (n = 113), (ii) base medium supplemented with 30 ng/ml Activin A (n = 115), (iii) base medium co-cultured with mouse embryonic fibroblast (n = 113), and (iv) base medium supplemented with 30 ng/ml Activin A and co-cultured with fibroblast (n = 115). Rate of growth, survivability, antrum formation, ovulation, embryonic development and steroid production were evaluated. Analysis of Variance and Duncan test were applied for analyzing. Results: Both co-culture and co-culture + Activin A groups showed significant difference (P<0.05) in growth (on days 4, 6, and 8 of culture period) and survival rates. However, there was no significant difference in antrum formation, ovulation rate, and embryonic development of ovulated oocytes. There were significant differences (P<0.05) in the estradiol production on days 8, 10, and 12 between co-culture + Activin A and the control group. Progesterone production also was significant (P<0.05) in co-culture + Activin A group on days 6, 8, 10, and 12 compared to control group. Conclusion: Fibroblast co-culture and Activin A promoted growth and survivability of preantral follicles. However, simultaneous use of them was more efficient. PMID:24375163

  20. Effect of GLY-HIS-LYS and its copper complex on TGF-β secretion in normal human dermal fibroblasts.

    PubMed

    Gruchlik, Arkadiusz; Chodurek, Ewa; Dzierzewicz, Zofia

    2014-01-01

    Transforming growth factor β (TGF-β) is a cytokine involved in a wide variety of biological process- es such as cell growth, differentiation and proliferation, apoptosis and regulation of the immune response. It has an important role in wound healing process, fibrosis and scar tissue formation. Similarly to TGF-β1, insulin growth factor (IGF) family is expressed locally in response to tissue injury. Treatment of dermal fibroblasts with IGF-1 caused a substantial induction of TGF-β1 mRNA. Not a great deal of research so far has focused on IGF-2. Much attention has been focused on the tripeptides such as Gly-His-Lys (GHK) and their copper complexes, which have a high activity and good skin tolerance. Recent data suggest that their physiological role has been related to the process of wound healing, tissue repair and skin inflammation. In the present study, the influence of 1 nM solutions of GHK, GHK-Cu and CuCl2, on IGF-2-dependent TGF-β1 secretion in normal human dermal fibroblasts cells was investigated. Fibroblasts were cultured in 24-well plates. Total TGF-β1 pro- tein was evaluated using the ELISA kit. The Bradford reagent was used to determine the total quantity of cel- lular protein. Treatment of fibroblasts with 100 ng/mL IGF-2 resulted in a significant increase in TGF-β1 secretion. GHK and its copper complex and free copper ions decreased IGF-2-dependent TGF-β1 secretion. Our observations provide some new information on the potential use of that peptide contained in cosmetics to treat and prevent the formation of hypertrophic scars. PMID:25745767

  1. Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: an in vitro study

    SciTech Connect

    Glassberg, E.; Lask, G.P.; Tan, E.M.; Uitto, J.

    1988-01-01

    The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells--erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including 1) viability of cells by trypan blue exclusion test; 2) cell proliferation by (3H)thymidine incorporation; and 3) rate of protein synthesis using (3H)leucine incorporation as a marker. In endothelial cell cultures, both (3H)thymidine and (3H)leucine incorporations were inhibited at energy levels of 5-12 J/cm2 (P less than 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (greater than or equal to 12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.

  2. Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells.

    PubMed

    Hati Boruah, Jyoti Lakshmi; Ranjan, Rakesh; Gogoi, Hamen; Pandey, Saurabh Kumar; Kumar, Dharmendra; Phukan, Amlan Jyoti; Bori, Joygeswar; Sarkhel, Bikash Chandra

    2016-01-01

    Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique. PMID:26690650

  3. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    SciTech Connect

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-03-05

    Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M and approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.

  4. Regulation of metalloproteinases and NF-kappaB activation in rabbit synovial fibroblasts via E prostaglandins and Erk: contrasting effects of nabumetone and 6MNA.

    PubMed

    Pillinger, Michael H; Dinsell, Victoria; Apsel, Beth; Tolani, Sonia N; Marjanovic, Nada; Chan, Edwin S L; Gomez, Paul; Clancy, Robert; Chang, Lih-Fan; Abramson, Steven B

    2004-07-01

    1 Nabumetone is a prodrug that is converted in vivo into 6-methoxy-2-naphthylacetic acid (6MNA), a cyclooxygenase inhibitor with anti-inflammatory properties. We tested the effects of nabumetone and 6MNA on the inflammatory responses of synovial fibroblasts (SFs). 2 Brief exposures to 6MNA (50-150 microm) had no effect on IL-1beta/TNF-alpha (each 20 ng ml(-1))-stimulated Erk activation. Longer exposures depleted prostaglandin E1 (PGE1) as much as 70%, and stimulated Erk as much as 300%. Nabumetone (150 microm) inhibited Erk activation by 60-80%. 6MNA (50-150 microm) stimulated (approximately 200%) and nabumetone (150 microm) inhibited (approximately 50%) matrix metalloproteinase (MMP)-1, but not MMP-13 secretion from SFs. 3 6MNA stimulation of MMP-1 secretion was inhibited approximately 30% by PGE1 (1 microm) and approximately 80% by the Erk pathway inhibitor UO126 (10 microm), confirming that PGE depletion and Erk activation mediate MMP-1 secretion by 6MNA. 4 Consistent with its role as an Erk inhibitor, nabumetone (150 microm) abrogated 6MNA enhancement of MMP-1 secretion. 5 UO126 (10 microm) and nabumetone (150 microm) inhibited (approximately 70 and 40%, respectively), but 6MNA (150 microm) enhanced (approximately 40%), NF-kappaB activation. 6 Our data indicate that 6MNA shares with other COX inhibitors several proinflammatory effects on synovial fibroblasts. In contrast, nabumetone demonstrates anti-inflammatory and potentially arthroprotective effects that have not been previously appreciated. PMID:15210577

  5. Differential Response and Priming Dose Effect on the Proteome of Human Fibroblast and Stem Cells Induced by Exposure to Low Doses of Ionizing Radiation.

    PubMed

    Hauptmann, Monika; Haghdoost, Siamak; Gomolka, Maria; Sarioglu, Hakan; Ueffing, Marius; Dietz, Anne; Kulka, Ulrike; Unger, Kristian; Babini, Gabriele; Harms-Ringdahl, Mats; Ottolenghi, Andrea; Hornhardt, Sabine

    2016-03-01

    It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation. PMID:26934482

  6. Effects of the Novel Compound DK223 ([1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) on Migration and Proliferation of Human Keratinocytes and Primary Dermal Fibroblasts

    PubMed Central

    Ho, Manh Tin; Kang, Hyun Sik; Huh, Jung Sik; Kim, Young Mee; Lim, Yoongho; Cho, Moonjae

    2014-01-01

    Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction. PMID:25056546

  7. A CXCL5- and bFGF-Dependent Effect of PDGF-B-Activated Fibroblasts in Promoting Trafficking and Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Nedeau, April E.; Bauer, Richard J.; Gallagher, Katherine; Chen, Haiying; Liu, Zhao-Jun

    2008-01-01

    Adult bone marrow-derived mesenchymal stem cells (MSCs) are able to differentiate into myofibroblasts and be recruited into wound lesions and contribute to wound healing. The cellular and molecular mechanism responsible for MSC trafficking and differentiation, however, are poorly understood. Local resting resident fibroblasts are activated after injury and play a critical role in recruiting MSCs. We investigated the role of platelet derived growth factor-B-activated fibroblasts (PDGF-B-aFBs) in regulating recruitment, migration and differentiation of MSCs from GFP transgenic mice in an in vitro wound healing assay and a novel three-dimensional (3D) model. PDGF-B-aFBs caused significant increases in MSCs migration velocity compared to control as demonstrated by time-lapse photography in an in vitro wound healing assay. Consistently, invasion/migration of MSCs into 3D collagen gels was enhanced in the presence of PDGF-B-aFbs. In addition, PDGF-B-aFBs induced differentiation of MSCs into myofibroblast. The regulatory effects of PDGF-B-aFBs are likely to be mediated by basic fibroblast growth factor (bFGF) and epithelial neutrophil activating peptide-78 (ENA-78 or CXCL5) as protein array analysis indicated an elevated levels of these two soluble factors in culture supernatant of PDGF-B-aFBs. Blocking antibodies against bFGF and CXCL5 were able to inhibit both trafficking and differentiation of MSCs into 3D collagen gels while supplement of exogenous bFGF and/or CXCL5 promoted invasion/migration of MSCs into 3D collagen gels. Our results reveal that PDGF-B-aFBs play a key role in recruitment/migration and differentiation of MSCs and implicate a bFGF- and CXCL5-dependent mechanism in mediating these effects. PMID:18570917

  8. Skin Permeabilization for Transdermal Drug Delivery: Recent Advances and Future Prospects

    PubMed Central

    Schoellhammer, Carl M.; Blankschtein, Daniel; Langer, Robert

    2014-01-01

    Introduction Transdermal delivery has potential advantages over other routes of administration. It could reduce first-pass metabolism associated with oral delivery and is less painful than injections. However, the outermost layer of the skin, the stratum corneum (SC), limits passive diffusion to small lipophilic molecules. Therefore, methods are needed to safely permeabilize the SC so that ionic and larger molecules may be delivered transdermally. Areas Covered This review focuses on low-frequency sonophoresis, microneedles, electroporation and iontophoresis, and combinations of these methods to permeabilize the SC. The mechanisms of enhancement and developments in the last five years are discussed. Potentially high-impact applications, including protein delivery, vaccination, and sensing, are presented. Finally, commercial interest and clinical trials are discussed. Expert Opinion Not all permeabilization methods are appropriate for all applications. Focused studies into applications utilizing the advantages of each method are needed. The total dose and kinetics of delivery must be considered. Vaccination is one application where permeabilization methods could make an impact. Protein delivery and analyte sensing are also areas of potential impact, although the amount of material that can be delivered (or extracted) is of critical importance. Additional work on the miniaturization of these technologies will help to increase commercial interest. PMID:24392787

  9. Fibroblast biology in pterygia.

    PubMed

    Kim, Kyoung Woo; Park, Soo Hyun; Kim, Jae Chan

    2016-01-01

    Activation of fibroblasts is a vital process during wound healing. However, if prolonged and exaggerated, profibrotic pathways lead to tissue fibrosis or scarring and further organ malfunction. Although the pathogenesis of pterygium is known to be multi-factorial, additional studies are needed to better understand the pathways initiated by fibroblast activation for the purpose of therapeutic translation. Regarding pterygium as a possible systemic disorder, we discuss the different cell types that pterygium fibroblasts originate from. These may include bone marrow-derived progenitor cells, cells undergoing epithelial-mesenchymal transition (EMT), and local resident stromal cells. We also describe how pterygium fibroblasts can be activated and perpetuate profibrotic signaling elicited by various proliferative drivers, immune-inflammation, and novel factors such as stromal cell-derived factor-1 (SDF-1) as well as a known key fibrotic factor, transforming growth factor-beta (TGF-β). Finally, epigenetic modification is discussed to explain inherited susceptibility to pterygium. PMID:26675401

  10. Contactless magneto-permeabilization for intracellular plasmid DNA delivery in-vivo

    PubMed Central

    Kardos, Thomas J.; Rabussay, Dietmar P.

    2012-01-01

    Electroporation, an attractive process for delivering DNA and other molecules into target cells in vivo and in vitro is limited by the necessity of electrodes that need to be in contact with the subject or object to be electroporated. We have used magnetic fields, which do not require material contact with the subject, to temporarily permeabilize cells in guinea pig skin in vivo to enhance uptake and expression of GFP plasmid DNA. The results show for the first time that magnetic fields can trigger a process likely similar to electroporation. In designing the magnetic pulses, our most important criterion was a high rate of change of the magnetic field, based on the principle described by Michael Faraday which is expressed by the formula: E = -dB/dt, (E, electric field, B, magnetic field, t, time). Magnetic fields were generated by a flat electromagnet in a hand-held applicator positioned above the target tissue. The magnetic pulses had a peak magnetic flux density of 4 tesla; 50 pulses were applied in 5 sec. Biphasic magnetic pulses were twice as effective as monophasic pulses and about equally effective as traditional electroporation pulses . Advantages of magnetopermeabilization over electoporation include: No contact between applicator and subject (“contact-less”); no need for invasive, disposable, sterile electrodes (“needle-less”); no pain from needles and reduced overall pain; no known side effects; easier and faster to administer than electroporation; less expensive due to absence of disposables; and, importantly, greater tissue penetration of the magnetic field allowing treatment of anatomical areas inaccessible by electroporation. PMID:22894955

  11. Contactless magneto-permeabilization for intracellular plasmid DNA delivery in-vivo.

    PubMed

    Kardos, Thomas J; Rabussay, Dietmar P

    2012-11-01

    Electroporation, an attractive process for delivering DNA and other molecules into target cells in vivo and in vitro is limited by the necessity of electrodes that need to be in contact with the subject or object to be electroporated. We have used magnetic fields, which do not require material contact with the subject, to temporarily permeabilize cells in guinea pig skin in vivo to enhance uptake and expression of GFP plasmid DNA. The results show for the first time that magnetic fields can trigger a process likely similar to electroporation. In designing the magnetic pulses, our most important criterion was a high rate of change of the magnetic field, based on the principle described by Michael Faraday which is expressed by the formula: E = -dB/dt, (E, electric field, B, magnetic field, t, time). Magnetic fields were generated by a flat electromagnet in a hand-held applicator positioned above the target tissue. The magnetic pulses had a peak magnetic flux density of 4 tesla; 50 pulses were applied in 5 sec. Biphasic magnetic pulses were twice as effective as monophasic pulses and about equally effective as traditional electroporation pulses . Advantages of magnetopermeabilization over electoporation include: No contact between applicator and subject ("contact-less"); no need for invasive, disposable, sterile electrodes ("needle-less"); no pain from needles and reduced overall pain; no known side effects; easier and faster to administer than electroporation; less expensive due to absence of disposables; and, importantly, greater tissue penetration of the magnetic field allowing treatment of anatomical areas inaccessible by electroporation. PMID:22894955

  12. A novel toxicity mechanism of CdSe nanoparticles to Saccharomyces cerevisiae: enhancement of vacuolar membrane permeabilization (VMP).

    PubMed

    Sun, Meiqing; Yu, Qilin; Liu, Ming; Chen, Xiaoyan; Liu, Zhe; Zhou, Hao; Yuan, Yingjin; Liu, Lu; Li, Mingchun; Zhang, Chengdong

    2014-09-01

    Cadmium selenide (CdSe) nanoparticles are implemented in a wide range of applications, but their potential risk to the ecosystem, especially to the organisms essential for the maintenance of ecosystem homeostasis, such as fungal populations, plants and bacteria, remains to be elucidated. In this study, we investigated their toxicity to one of the most important fungal model organisms, Saccharomyces cerevisiae. Growth inhibition assays revealed that the synthesized CdSe nanoparticles with the sizes of 20-30 nm had strong inhibitory effect on yeast growth (IC50=80 ppm). This toxicity was not attributed to mitochondrial dysfunction and autophagy, but was dependent on End3-mediated endocytosis, and was associated with reactive oxygen species (ROS) accumulation and an enhancement of vacuolar membrane permeabilization (VMP). These results reveal a key role of the vacuole during the interaction between CdSe nanoparticles and yeast cells. PMID:25014418

  13. The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

    PubMed

    Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

    2016-03-01

    It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded

  14. Effect of 405-nm high-intensity narrow-spectrum light on fibroblast-populated collagen lattices: an in vitro model of wound healing

    NASA Astrophysics Data System (ADS)

    McDonald, Richard; MacGregor, Scott J.; Anderson, John G.; Maclean, Michelle; Grant, M. Helen

    2011-04-01

    High-intensity narrow-spectrum (HINS) 405-nm light is a novel technology developed to address the significant problem of health-care associated infection. Its potential for wound-decontamination applications is assessed on mammalian cells and bacteria. The fibroblast-populated collagen lattice (FPCL) is used as an in vitro model of wound healing, and the effect of HINS light on contraction is examined. Effects on cell proliferation, morphological changes, and α-smooth muscle actin (α-SMA) expression are investigated. Bactericidal effects are assessed using the bacterium Staphylococcus epidermidis. Low doses of HINS light were found to have no significant inhibitory effects on FPCL contraction, cell proliferation, or α-SMA expression. Doses of up to 18 Jcm-2 had no significant inhibitory effects on FPCL cell numbers, and this dose was shown to cause almost complete inactivation of bacteria. These results show that HINS light has potential for disinfection applications without adversely influencing wound healing.

  15. Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

    PubMed

    Blaimauer, Karin; Watzinger, Elisabeth; Erovic, Boban M; Martinek, Helga; Jagersberger, Tamara; Thurnher, Dietmar

    2006-01-01

    Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct. PMID:16804300

  16. Biostimulative effects of Nd:YAG Q-switch dye on normal human fibroblast cultures: study of a new chemosensitizing agent for the Nd:YAG laser

    SciTech Connect

    Castro, D.J.; Saxton, R.E.; Fetterman, H.R.; Castro, D.J.; Ward, P.H.

    1987-12-01

    Kodak Q-switch II is a new chemical with an absorption maxima at 1051 nm, designed to be used as an Nd:YAG dye laser. The potential for this dye as a new chemosensitizing agent in the treatment of connective tissue diseases and wound healing with low energy Nd:YAG laser was examined. Two normal fibroblast cell lines were tested for sensitivity to various levels of this dye in vitro. These cells were exposed to Q-switch II dye at concentrations of 0.01, 0.1, 1, 10, 50, and 100 micrograms/ml for 1 and 24 hours. Cell viability was assessed by the trypan blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of (/sup 3/H)-thymidine at 6 and 24 hours postexposure to Q-switch II dye. At concentrations up to 10 micrograms/ml, both cell lines tested showed no changes in cell viability. However, at concentrations equal or higher than 50 micrograms/ml, more than 40% of the fibroblasts incorporated trypan blue after 24 hours of exposure to this dye, indicating significant cell destruction. The results indicate that Q-switch II dye is nontoxic to normal human fibroblast cultures and showed significant biostimulative effects on cell duplication at concentrations equal to or lower than 10 micrograms/ml. Further studies will be required to determine the usefulness of Q-switch II dye as a new photochemosensitizing agent for potential biostimulation of wound healing and/or treatment of connective tissue diseases with the Nd:YAG laser (near infrared, 1060 nm) at nonthermal levels of energies.

  17. Redox-Active Profile Characterization of Remirea maritima Extracts and Its Cytotoxic Effect in Mouse Fibroblasts (L929) and Melanoma (B16F10) Cells.

    PubMed

    Dória, Grace Anne A; Santos, Anderson R; Bittencourt, Leonardo S; Bortolin, Rafael C; Menezes, Paula P; Vasconcelos, Bruno S; Souza, Rebeca O; Fonseca, Maria José V; Santos, Alan Diego C; Saravanan, Shanmugam; Silva, Francilene A; Gelain, Daniel P; Moreira, José Cláudio F; Prata, Ana Paula N; Quintans-Júnior, Lucindo J; Araújo, Adriano A S

    2015-01-01

    Remirea maritima is a tropical plant with a reticulated root system belonging to the family Cyperaceae, also known to have biologically active secondary metabolites. However, very few data on R. maritima's biological actions are available and there are no reports regarding the redox-active profile of this plant. In this study, we examined the total phenolic content of Remirea maritima hydroalcoholic (RMHA) extracts, redox properties against different reactive species generated in vitro and their cytotoxic effect against fibroblasts (L929) and melanoma (B16F10) cells. Total reactive antioxidant potential index (TRAP) and total antioxidant reactivity (TAR) results revealed that RMHA at all concentrations tested showed significant antioxidant capacity. RMHA was also effective against hydroxyl radical formation, reduction of Fe3+ to Fe2+ and in scavenging nitric oxide (NO) radicals. In vitro, the level of lipid peroxidation was reduced by RMHA extract and the data showed significant oxidative damage protection. The RMHA cytotoxicity was evaluated by a neutral red assay in fibroblast (L929) and melanome (B16F10) cells. The obtained results showed that the RMHA (40 and 80 µg/mL, respectively) reduced 70% of the viable cells. In conclusion, this study represents the first report regarding the antioxidant and anti-proliferative potential of R. maritima against B16F10 melanoma cells. PMID:26121396

  18. Expression and Functional Characterization of Bluetongue Virus VP5 Protein: Role in Cellular Permeabilization

    PubMed Central

    Hassan, S. H.; Wirblich, C.; Forzan, M.; Roy, P.

    2001-01-01

    Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis. PMID:11507181

  19. Hydrogen sulphide inhibits carbachol-induced contractile responses in β-escin permeabilized guinea-pig taenia caecum.

    PubMed

    Denizalti, Merve; Durlu-Kandilci, N Tugba; Bozkurt, T Emrah; Sahin-Erdemli, Inci

    2011-05-11

    Hydrogen sulphide (H(2)S) is an endogenous mediator producing a potent relaxation response in vascular and non-vascular smooth muscles. While ATP-sensitive potassium channels are mainly involved in this relaxant effect in vascular smooth muscle, the mechanism in other smooth muscles has not been revealed yet. In the present study, we investigated how H(2)S relaxes non-vascular smooth muscle by using intact and β-escin permeabilized guinea-pig taenia caecum. In intact tissues, concentration-dependent relaxation response to H(2)S donor NaHS in carbachol-precontracted preparations did not change in the presence of a K(ATP) channel blocker glibenclamide, adenylate cyclase inhibitor SQ-22536, guanylate cyclase inhibitor ODQ, protein kinase A inhibitor KT-5720, protein kinase C inhibitor H-7, tetrodotoxin, apamin/charybdotoxin, NOS inhibitor L-NAME and cyclooxygenase inhibitor indomethacin. We then studied how H(2)S affected carbachol- or Ca(2+)-induced contractions in permeabilized tissues. When Ca(2+) was clamped to a constant value (pCa6), a further contraction could be elicited by carbachol that was decreased by NaHS. This decrease in contraction was reversed by catalase but not by superoxide dismutase or N-acetyl cysteine. The sarcoplasmic reticulum Ca(2+)-ATPase pump inhibitor, cyclopiazonic acid, also decreased the carbachol-induced contraction that was further inhibited by NaHS. Mitochondrial proton pump inhibitor carbonyl cyanide p-trifluromethoxyphenylhydrazone also decreased the carbachol-induced contraction but this was not additionally changed by NaHS. The carbachol-induced Ca(2+) sensitization, calcium concentration-response curves, IP(3)- and caffeine-induced contractions were not affected by NaHS. In conclusion, we propose that hydrogen peroxide and mitochondria may have a role in H(2)S-induced relaxation response in taenia caecum. PMID:21371473

  20. Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

    SciTech Connect

    Randazzo, P.A.; Morey, V.A.; Polishook, A.K.; Jarett, L. )

    1990-09-01

    The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle.

  1. Effect of Gly-Gly-His, Gly-His-Lys and their copper complexes on TNF-alpha-dependent IL-6 secretion in normal human dermal fibroblasts.

    PubMed

    Gruchlik, Arkadiusz; Jurzak, Magdalena; Chodurek, Ewa; Dzierzewicz, Zofia

    2012-01-01

    Cosmeceuticals represent a marriage between cosmetics and pharmaceuticals. There are numerous cosmeceutically active products which can be broadly classified into the following categories: antioxidants, oligopeptides, growth factors and pigment lightning agents. Much attention has been focused on the tripeptides such as Gly-His-Lys (GHK) and Gly-Gly-His (GGH) and their copper complexes, which have a high activity and good skin tolerance. Recent data suggested their physiological role in process of wound healing, tissue repair and skin inflammation. The mechanism of anti-inflammatory properties of these peptides is not clear. The aim of the study was evaluation of influence of two peptides GGH. GHK and their copper complexes and saccharomyces/copper ferment (Oligolides Copper) on secretion of pro-inflammatory IL-6 in normal human dermal fibroblasts NHDF cell line. IL-6 was evaluated using the ELISA kit. GGH, GHK, CuCl2 and their copper complexes decreased TNF-alpha-dependent IL-6 secretion in fibroblasts. IL-6 is crucial for normal wound healing, skin inflammation and UVB-induced erythema. Because of the anti-inflammatory properties, the copper-peptides could be used on the skin surface instead of corticosteroids or non-steroidal anti-inflammatory drugs, which have more side effects. Our observations provide some new information about the role of these tripeptides in skin inflammation. PMID:23285694

  2. Evaluation of the effect of laser radiation on fibroblast proliferation in repair of skin wounds of rats with iron deficiency anemia

    NASA Astrophysics Data System (ADS)

    DeCastro, Isabele C. V.; Oliveira-Sampaio, Susana C. P.; Monteiro, Juliana S. de C.; Ferreira, Maria de Fátima L.; Cangussu, Maria T.; N. dos Santos, Jean; Pinheiro, Antonio Luiz B.

    2011-03-01

    The aim of this study was to assess the effect of low- level laser therapy (LLLT) on fibroblast proliferation on wound repair of rats with Iron deficiency anemia since there is no reports on literature about this subject. Iron deficiency anemia was induced on 36 newborn rats then an excisional wound was created on the dorsum of the animals which were divided into four groups: (I) - non-anemic, (II) - Anemic, (III) - non-anemic + LLLT, (IV) Anemic+ LLLT. The animals in each group were sacrificed at 7, 14 and 21 days. Laser irradiation was performed on each group (λ660nm,40Mw,CW) by contact mode with a dose of 2,5J/ cm2 in four points on the area of the wound and total of 10J/cm2 per session. Data were evaluated by analysis of variance (ANOVA) followed by Paired t-test. The results showed LLLT was able to stimulate fibroblastic proliferation in rats with iron deficiency anemia at the 21st day while at control group (III) no statistically significant differences was found.

  3. Dupuytren's Contracture: Fibroblast Contraction?

    PubMed Central

    Gabbiani, Giulio; Majno, Guido

    1972-01-01

    In 6 cases of Dupuytren's disease and 1 of Ledderhose's disease, the nodules of the palmar and plantar aponeurosis were examined by light and electron microscopy. The cells composing these nodules, presumably fibroblasts, showed three significant ultrastructural features: (1) a fibrillar system similar to that of smooth muscle cells; (2) nuclear deformations such as are found in contracted cells, the severest being recognizable by light microscopy (cross-banded nuclei); (3) cell-to-cell and cell-to-stroma attachments. Based on these data and on recent information about the biology of the fibroblasts, it is suggested that these cells are fibroblasts that have modulated into contractile cells (myofibroblasts), and that their contraction plays a role in the pathogenesis of the contracture observed clinically. ImagesFig 10Fig 5Fig 11Fig 6 and 7Fig 8Fig 1Fig 2Fig 9Fig 3Fig 4 PMID:5009249

  4. The effect of MTAD, an endodontic irrigant, on fibroblast attachment to periodontally affected root surfaces: A SEM analysis

    PubMed Central

    Ghandi, Mostafa; Houshmand, Behzad; Nekoofar, Mohammad H.; Tabor, Rachel K.; Yadeghari, Zahra; Dummer, Paul M. H.

    2013-01-01

    Background: Root surface debridement (RSD) is necessary to create an environment suitable for reattachment of the periodontium. Root surface conditioning may aid the formation of a biocompatible surface suitable for cell reattachment. BioPure™ MTAD (mixture of Doxycycline, citric acid and a detergent) is an endodontic irrigant with antibacterial properties and the ability to remove smear layer. It was hypothesized that MTAD may be useful for root surface conditioning. The efficacy of MTAD as a conditioner was measured by examining fibroblast attachment to root surfaces. Materials and Methods: Thirty-two specimens of human teeth with advanced periodontal disease were used. The surfaces were root planed until smooth. Half of the specimens were treated with 0.9% saline and the other samples with Biopure MTAD. As a negative control group, five further samples were left unscaled with surface calculus. Human gingival fibroblast cells HGF1-PI1 were cultured and poured over the tooth specimens and incubated. After fixation, the samples were sputter-coated with gold and examined with a SEM. The morphology and number of attached, fixed viable cells were examined. The data was analysed using the Mann-Whitney-U statistical test. Results: There was no significant difference between the numbers of attached cells in the experimental group treated with MTAD and the control group treated with saline. Little or no attached cells were seen in the negative control group. Conclusion: RSD created an environment suitable for cell growth and attachment in a laboratory setting. The use of MTAD did not promote the attachment and growth of cells on the surface of human roots following RSD. PMID:23869124

  5. Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

    PubMed Central

    Senevirathne, Mahinda; Kim, Soo-Hyun

    2010-01-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro. PMID:20607062

  6. The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts.

    PubMed

    Korade, Zeljka; Kim, Hye-Young H; Tallman, Keri A; Liu, Wei; Koczok, Katalin; Balogh, Istvan; Xu, Libin; Mirnics, Karoly; Porter, Ned A

    2016-02-11

    Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway. PMID:26789657

  7. Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

    SciTech Connect

    Stępnik, Maciej; Arkusz, Joanna; Smok-Pieniążek, Anna; Bratek-Skicki, Anna; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A.; Gromadzińska, Jolanta; De Jong, Wim H.; Rydzyński, Konrad

    2012-08-15

    The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but not to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows

  8. Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide

    PubMed Central

    Demir, Erhan; Hoffmanns, Martin A.; Baldus, Sabrina; Fuchs, Paul C.; Awakowicz, Peter; Suschek, Christoph V.; Opländer, Christian

    2015-01-01

    The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well

  9. Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide.

    PubMed

    Balzer, Julian; Heuer, Kiara; Demir, Erhan; Hoffmanns, Martin A; Baldus, Sabrina; Fuchs, Paul C; Awakowicz, Peter; Suschek, Christoph V; Opländer, Christian

    2015-01-01

    The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal "cold" atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60-300 s; ~77-66%) or exposure to freshly DBD-treated PBS (60-300 s; ~75-55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in

  10. Compact Electro-Permeabilization System for Controlled Treatment of Biological Cells and Cell Medium Conductivity Change Measurement

    NASA Astrophysics Data System (ADS)

    Novickij, Vitalij; Grainys, Audrius; Novickij, Jurij; Tolvaisiene, Sonata; Markovskaja, Svetlana

    2014-10-01

    Subjection of biological cells to high intensity pulsed electric field results in the permeabilization of the cell membrane. Measurement of the electrical conductivity change allows an analysis of the dynamics of the process, determination of the permeabilization thresholds, and ion efflux influence. In this work a compact electro-permeabilization system for controlled treatment of biological cells is presented. The system is capable of delivering 5 μs - 5 ms repetitive square wave electric field pulses with amplitude up to 1 kV. Evaluation of the cell medium conductivity change is implemented in the setup, allowing indirect measurement of the ion concentration changes occurring due to the cell membrane permeabilization. The simulation model using SPICE and the experimental data of the proposed system are presented in this work. Experimental data with biological cells is also overviewed

  11. Induction of Highly Curved Structures in Relation to Membrane Permeabilization and Budding by the Triterpenoid Saponins, α- and δ-Hederin

    PubMed Central

    Lorent, Joseph; Le Duff, Cécile S.; Quetin-Leclercq, Joelle; Mingeot-Leclercq, Marie-Paule

    2013-01-01

    The interactions of triterpenoid monodesmosidic saponins, α-hederin and δ-hederin, with lipid membranes are involved in their permeabilizing effect. Unfortunately, the interactions of these saponins with lipid membranes are largely unknown, as are the roles of cholesterol or the branched sugar moieties (two for α-hederin and one for δ-hederin) on the aglycone backbone, hederagenin. The differences in sugar moieties are responsible for differences in the molecular shape of the saponins and the effects on membrane curvature that should be the most positive for α-hederin in a transbilayer direction. In large unilamellar vesicles and monocyte cells, we showed that membrane permeabilization was dependent on the presence of membrane cholesterol and saponin sugar chains, being largest for α-hederin and smallest for hederagenin. In the presence of cholesterol, α-hederin induced the formation of nonbilayer phases with a higher rate of Brownian tumbling or lateral diffusion. A reduction of Laurdan's generalized polarization in relation to change in order of the polar heads of phospholipids was observed. Using giant unilamellar vesicles, we visualized the formation of wrinkled borders, the decrease in liposome size, budding, and the formation of macroscopic pores. All these processes are highly dependent on the sugars linked to the aglycone, with α-hederin showing a greater ability to induce pore formation and δ-hederin being more efficient in inducing budding. Hederagenin induced intravesicular budding but no pore formation. Based on these results, a curvature-driven permeabilization mechanism dependent on the interaction between saponin and sterols and on the molecular shape of the saponin and its ability to induce local spontaneous curvature is proposed. PMID:23530040

  12. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis

    PubMed Central

    Salabei, Joshua K.; Gibb, Andrew A.; Hill, Bradford G.

    2014-01-01

    Extracellular flux (XF) analysis has become a mainstream method to measure bioenergetic function in cells and tissues. While this technique is commonly used to measure energetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin, digitonin, or recombinant perfringolysin O (XF PMP reagent) and provided with specific substrates to measure complex I- or II-mediated respiratory activity, Complex III+IV respiratory activity, or Complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid oxidation or glutamine oxidation, respectively. This protocol allows for such measurements using a minimal number of cells compared with other protocols, without the need for mitochondrial isolation. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, following preparative steps, takes approximately 6 hours to complete. PMID:24457333

  13. Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions.

    PubMed

    Bansal-Mutalik, Ritu; Gaikar, Vilas G

    2003-01-01

    Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible. PMID:14656146

  14. Yeast cell mortality related to a high-pressure shift: occurrence of cell membrane permeabilization.

    PubMed

    Perrier-Cornet, J M; Hayert, M; Gervais, P

    1999-07-01

    The shrinkage of yeast cells caused by high-pressure treatment (250 MPa, 15 min) was investigated using direct microscopic observation. A viable staining method after treatment allowed the volume variation of two populations to be distinguished: an irreversible volume decrease (about 35% of the initial volume) of pressure-inactivated cells during pressure holding time, and viable cells, which were less affected. A mass transfer was then induced during high-pressure treatment. Causes of this transfer seem to be related to a pressure-induced membrane permeabilization, allowing a subsequent leakage of internal solutes, where three ions (Na+, K+ and Ca2+), plus endogenous glycerol, were verified. This glycerol leakage was found to occur after yeast pressurization in a medium having low water activity, although the yeast was not inactivated. All these observations lead to the hypothesis that pressure-induced cell permeabilization could be the cause of yeast inactivation under pressure. PMID:10432582

  15. Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

    PubMed Central

    Menestrina, G; Pederzolli, C; Forti, S; Gambale, F

    1991-01-01

    We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin. Images FIGURE 7 FIGURE 8 FIGURE 12

  16. Saponin-permeabilization is not a viable alternative to isolated mitochondria for assessing oxidative metabolism in hibernation

    PubMed Central

    Mathers, Katherine E.; Staples, James F.

    2015-01-01

    ABSTRACT Saponin permeabilization of tissue slices is increasingly popular for characterizing mitochondrial function largely because it is fast, easy, requires little tissue and leaves much of the cell intact. This technique is well described for mammalian muscle and brain, but not for liver. We sought to evaluate how saponin permeabilization reflects aspects of liver energy metabolism typically assessed in isolated mitochondria. We studied the ground squirrel (Ictidomys tridecemlineatus Mitchell), a hibernating mammal that shows profound and acute whole-animal metabolic suppression in the transition from winter euthermia to torpor. This reversible metabolic suppression is also reflected in the metabolism of isolated liver mitochondria. In this study we compared euthermic and torpid animals using saponin permeabilized tissue and mitochondria isolated from the same livers. As previously demonstrated, isolated mitochondria have state 3 respiration rates, fueled by succinate, that are suppressed by 60-70% during torpor. This result holds whether respiration is standardized to mitochondrial protein, cytochrome a content or citrate synthase activity. In contrast, saponin-permeabilized liver tissue, show no such suppression in torpor. Neither citrate synthase activity nor VDAC content differ between torpor and euthermia, indicating that mitochondrial content remains constant in both permeabilized tissue and isolated mitochondria. In contrast succinate dehydrogenase activity is suppressed during torpor in isolated mitochondria, but not in permeabilized tissue. Mechanisms underlying metabolic suppression in torpor may have been reversed by the permeabilization process. As a result we cannot recommend saponin permeabilization for assessing liver mitochondrial function under conditions where acute changes in metabolism are known to occur. PMID:25979709

  17. Saponin-permeabilization is not a viable alternative to isolated mitochondria for assessing oxidative metabolism in hibernation.

    PubMed

    Mathers, Katherine E; Staples, James F

    2015-01-01

    Saponin permeabilization of tissue slices is increasingly popular for characterizing mitochondrial function largely because it is fast, easy, requires little tissue and leaves much of the cell intact. This technique is well described for mammalian muscle and brain, but not for liver. We sought to evaluate how saponin permeabilization reflects aspects of liver energy metabolism typically assessed in isolated mitochondria. We studied the ground squirrel (Ictidomys tridecemlineatus Mitchell), a hibernating mammal that shows profound and acute whole-animal metabolic suppression in the transition from winter euthermia to torpor. This reversible metabolic suppression is also reflected in the metabolism of isolated liver mitochondria. In this study we compared euthermic and torpid animals using saponin permeabilized tissue and mitochondria isolated from the same livers. As previously demonstrated, isolated mitochondria have state 3 respiration rates, fueled by succinate, that are suppressed by 60-70% during torpor. This result holds whether respiration is standardized to mitochondrial protein, cytochrome a content or citrate synthase activity. In contrast, saponin-permeabilized liver tissue, show no such suppression in torpor. Neither citrate synthase activity nor VDAC content differ between torpor and euthermia, indicating that mitochondrial content remains constant in both permeabilized tissue and isolated mitochondria. In contrast succinate dehydrogenase activity is suppressed during torpor in isolated mitochondria, but not in permeabilized tissue. Mechanisms underlying metabolic suppression in torpor may have been reversed by the permeabilization process. As a result we cannot recommend saponin permeabilization for assessing liver mitochondrial function under conditions where acute changes in metabolism are known to occur. PMID:25979709

  18. A Lack of Synergy Between Membrane-permeabilizing Cationic Antimicrobial Peptides and Conventional Antibiotics

    PubMed Central

    He, Jing; Starr, Charles G.; Wimley, William C.

    2014-01-01

    The rapid rise in morbidity and mortality from drug-resistant pathogenic bacteria has generated elevated interest in combination therapy using antimicrobial agents. Antimicrobial peptides (AMPs) are a candidate drug class to advance the development of combination therapies. Although the literature is ambiguous, the generic membrane disrupting activity of AMPs could enable them to synergize with conventional small molecule antibiotics by increasing access to the cell and by triggering membrane damage mediators. We used a novel assay to measure interactions, expressed as fractional inhibitory concentration (FIC), between four conventional antibiotics in combination with four well-characterized, membrane permeabilizing AMPs, against three species of Gram negative and Gram positive bacteria, giving 40 total pair-wise measurements of FIC with statistical uncertainties. We chose a set of AMPs that are known to dramatically disrupt the membranes of both Gram negative and Gram positive bacteria. Yet none of the membrane permeabilizing antimicrobial peptides interacted synergistically with any of the conventional antibiotic drugs in any organism. Large-scale membrane disruption and permeabilization by AMPs is not sufficient to drive them to act synergistically with chemical antibiotics in either Gram negative or Gram positive microbes. PMID:25268681

  19. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    PubMed

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

  20. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

    PubMed

    Salabei, Joshua K; Gibb, Andrew A; Hill, Bradford G

    2014-02-01

    Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I- or complex II-mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6-8 h to complete. PMID:24457333

  1. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.

    PubMed

    Freire, João Miguel; Gaspar, Diana; de la Torre, Beatriz Garcia; Veiga, Ana Salomé; Andreu, David; Castanho, Miguel A R B

    2015-02-01

    Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria. PMID:25445678

  2. A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

    PubMed Central

    Rand, Matthew D.

    2014-01-01

    The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules. PMID:25046169

  3. A new technique for reversible permeabilization of live cells for intracellular delivery of quantum dots

    NASA Astrophysics Data System (ADS)

    Medepalli, Krishnakiran; Alphenaar, Bruce W.; Keynton, Robert S.; Sethu, Palaniappan

    2013-05-01

    A major challenge with the use of quantum dots (QDs) for cellular imaging and biomolecular delivery is the attainment of QDs freely dispersed inside the cells. Conventional methods such as endocytosis, lipids based delivery and electroporation are associated with delivery of QDs in vesicles and/or as aggregates that are not monodispersed. In this study, we demonstrate a new technique for reversible permeabilization of cells to enable the introduction of freely dispersed QDs within the cytoplasm. Our approach combines osmosis driven fluid transport into cells achieved by creating a hypotonic environment and reversible permeabilization using low concentrations of cell permeabilization agents like Saponin. Our results confirm that highly efficient endocytosis-free intracellular delivery of QDs can be accomplished using this method. The best results were obtained when the cells were treated with 50 μg ml-1 Saponin in a hypotonic buffer at a 3:2 physiological buffer:DI water ratio for 5 min at 4 ° C.

  4. PKC activation increases Ca2+ sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms

    PubMed Central

    Dougherty, Patrick J.; Nepiyushchikh, Zhanna V.; Chakraborty, Sanjukta; Wang, Wei; Davis, Michael J.; Zawieja, David C.

    2014-01-01

    The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca2+. It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca2+. To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca2+ concentration, and the Ca2+-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca2+ sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca2+ sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser19, 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr38, or caldesmon at Ser789. Thus, these data indicate that PKC-dependent Ca2+ sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s). PMID:24414065

  5. Novel Nitrobenzazolo[3,2-a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Changes, and Mitochondrial Permeabilization

    PubMed Central

    Vélez, Christian; Cox, Osvaldo; Rosado-Berrios, Carlos A.; Molina, Dennise; Arroyo, Luz; Carro, Sujey; Filikov, Anton; Kumar, Vineet; Malhotra, Sanjay V.; Cordero, Marisol; Zayas, Beatriz

    2014-01-01

    This study reports the capacity of three nitro substituted benzazolo[3,2-a]quinolinium salts NBQs: NBQ 95 (NSC-763304), NBQ 38 (NSC 763305), and NBQ 97 (NSC-763306) as potential antitumor agents. NBQ’s are unnatural alkaloids possessing a positive charge that could facilitate interaction with cell organelles. The anticancer activities of these compounds were evaluated through the National Cancer Institute (NCI) 60 cell line screening which represents diverse histologies. The screening was performed at 10 µM on all cell lines. Results from the NCI screening indicated cytotoxicity activity on six cell lines. In order to explore a possible mechanism of action, a detailed biological activity study of NBQ 95 and NBQ 38 was performed on A431 human epidermoid carcinoma cells to determine an apoptotic pathway involving, cell cycle changes, DNA fragmentation, mutations, mitochondrial membrane permeabilization and caspases activation. DNA fragmentation, cell cycle effects, mutagenesis, mitochondrial permeabilization and activation of caspases were determined by fluorimetry and differential imaging. Our data showed that A431 growth was inhibited with an average IC50 of 30 µM. In terms of the mechanism, these compounds interacted with DNA causing fragmentation and cell cycle arrest at sub G0/G1 stage. Mutagenesis was higher for NBQ 38 and moderate for NBQ 95 Mitochon-drial permeabilization was observed with NBQ 38 and slightly for NBQ 95. Both compounds caused activation of Caspases 3 and 7 suggesting an apoptotic cell death pathway through an intrinsic mechanism. This study reports evidence of the toxicity of these novel compounds with overlapping structural and mechanistic similarities to ellipticine, a known anti-tumor compound. PMID:25243104

  6. Isolation, growth and differentiation of equine mesenchymal stem cells: effect of donor, source, amount of tissue and supplementation with basic fibroblast growth factor.

    PubMed

    Colleoni, Silvia; Bottani, Emanuela; Tessaro, Irene; Mari, Gaetano; Merlo, Barbara; Romagnoli, Noemi; Spadari, Alessandro; Galli, Cesare; Lazzari, Giovanna

    2009-12-01

    Mesenchymal stem cells (MSC) are increasingly used as therapeutical aid for the orthopaedic injuries in the horse. MSC populate different tissues but the most commonly used for clinical purposes are isolated from bone marrow or adipose tissue. The first objective of this study was to investigate if the donor animal, the tissue of origin and the technique of isolation could influence the number of MSC available for transplantation after a short-term expansion. The second aim was to devise a culture system capable of increasing MSC lifespan and we tested the effect of basic fibroblast growth factor (bFGF). Results indicate that MSC can be efficiently isolated from both sources and supplementation of bFGF enhances proliferation rate maintaining differentiation potential. In addition, this study shows that collection, expansion and storage of frozen MSC can be performed for later therapeutic use. PMID:19472068

  7. Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts, TIG-1, after freezing and thawing.

    PubMed

    Kondo, H; Yamamoto, K

    1981-06-01

    The viability and the recovery (cell attachment to the dish) after thawing of human diploid fibroblasts (TIG-1) frozen by four different methods were studied at different passages. Improved results were observed in a medium of 30% fetal bovine serum plus 15% glycerol, compared with the conventional medium which contained 10% fetal bovine serum plus 10% glycerol. Centrifugation to remove glycerol immediately after thawing had a negative effect on the viability and recovery of cells. The recovery of cells after freezing and thawing showed a maximal value in the middle of phase II (PD 35) during the finite lifespan of the cell (average PD 67). This results indicates that the cells at early and late passages are sensitive to injury by freezing and thawing. The modified method yielded improved recovery, especially in the cells at early and late passages, except for the extremely senile stage. The recovery was also affected by the state of cell growth after inoculation. PMID:7266075

  8. Characteristics of inositol trisphosphate mediated Ca/sup 2 +/ release from permeabilized hepatocytes

    SciTech Connect

    Joseph, S.K.; Williamson, J.R.

    1986-05-01

    Ca/sup 2 +/ release triggered by inositol trisphosphate (IP/sub 3/) has been measured in saponin-permeabilized hepatocytes with /sup 45/Ca/sup 2 +/ or Quin 2. The initial rate of Ca/sup 2 +/ release was not markedly affected by the incubation temperature (175 +/- 40 pmol/s/mg at 30/sup 0/C versus 133 +/- 24 pmol/s/mg at 4/sup 0/C). This result is consistent with the membrane translocation of Ca/sup 2 +/ occurring through an ion-channel rather than an ion-carrier. The amount of Ca/sup 2 +/ released by IP/sub 3/ was not affected by pH (6.5-8.0) or by compounds that inhibit voltage-gated Ca/sup 2 +/ channels. La/sup 3 +/ (100 ..mu..M) markedly inhibits the effect of 1 ..mu..M IP/sub 3/. The possibility that La/sup 3 +/ chelates IP/sub 3/ cannot be excluded since the effect of La/sup 3 +/ can be overcome by increasing the IP/sub 3/ concentration. IP/sub 3/-mediated Ca/sup 2 +/ release displays a requirement for a permeant cation in the incubation medium. Optimal release is observed with K/sup +/ gluconate. Other monovalent cations, with the exception of Li/sup +/, can substitute for K/sup +/. Permeant anions, at concentrations above 40 mM, inhibit Ca/sup 2 +/ release produced by IP/sub 3/. Cl/sup -/, Br/sup -/, I/sup -/, and SO/sub 4//sup 2 -/ were equally effective. Ca/sup 2 +/ release was not inhibited by DIDS or Furosemide. /sup 85/Sr/sup 2 +/ and /sup 54/Mn/sup 2 +/ fluxes were also stimulated by IP/sub 3/. These results suggest that IP/sub 3/ acts to gate a divalent cation channel. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membrane.

  9. Dynamic monitoring of GPER-mediated estrogenic effects in breast cancer associated fibroblasts: An alternative role of estrogen in mammary carcinoma development.

    PubMed

    Luo, Haojun; Liu, Manran; Luo, Shujuan; Yu, Tenghua; Wu, Chengyi; Yang, Guanglun; Tu, Gang

    2016-08-01

    Cancer associated fibroblasts (CAFs) are crucial contributors to breast cancer development. Estrogen affects mammary stroma in both physiological and pathophysiological conditions. We show here that estrogen (G-protein coupled) receptor (GPER) could be detected by immunohistochemistry in stromal fibroblasts of primary breast cancers. The presence of GPER expression was further confirmed by immunofluorescence and quantitative PCR in CAFs isolated from primary breast cancers. Based on dynamic monitoring by real time cell analyzer (RTCA) system, 17-β-estradiol (E2) as well as GPER specific agonist G1 were observed to trigger transient cell index increasing within an hour in a dosage-dependent manner in breast CAFs. In addition, E2 and G1 stimulated intracellular calcium modulation and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 within seconds and minutes in CAFs, respectively. Moreover, E2 and G1 promoted cell proliferation of breast CAFs measured by RTCA monitoring, cell viability assay and cell cycle analysis, and this promotion could be blocked by a GPER-selective antagonist G15. Interestingly, dynamic RTCA monitoring indicated that E2 increased adhesion of resuspended cells, and microscopy confirmed that E2 stimulated cell spreading. Both the adhesion and spreading were proposed to be mediated by GPER, since G1 also stimulated these effects similar to E2, and G15 reduced them. Moreover, GPER was found to mediate migration that was increased by E2 and G1 but reduced by G15 in RTCA cell migration assay and transwell assay. Accordingly, GPER mediates not only rapid actions but also slow effects including adhesion/spreading, proliferation and migration in breast CAFs. Estrogen is likely to affect tumor associated stroma and contributes to mammary carcinoma development through CAFs. PMID:27016131

  10. Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion, proliferation, migration and invasion of colon cancer cells.

    PubMed

    Dowling, Catríona M; Herranz Ors, Carmen; Kiely, Patrick A

    2014-01-01

    Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells. PMID:24935351

  11. Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells

    PubMed Central

    YANG, HUA; QIU, YING; ZENG, XIANGHUI; DING, YAN; ZENG, JIANYE; LU, KEHUAN; LI, DONGSHENG

    2016-01-01

    The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×108 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1. PMID:27313670

  12. The Effect of MSCs Derived from the Human Umbilical Cord Transduced by Fibroblast Growth Factor-20 on Parkinson's Disease

    PubMed Central

    Jinfeng, Li; Yunliang, Wang; Xinshan, Liu; Shanshan, Wang; Chunyang, Xu; Peng, Xue; Xiaopeng, Yang; Zhixiu, Xu; Honglei, Yin; Xia, Cao; Haifeng, Duan; Bingzhen, Cao

    2016-01-01

    Cell therapy is a potential therapeutic approach for Parkinson's disease (PD). Mesenchymal stem cells derived from the human umbilical cord (hUC-MSCs) give priority to PD patients because of multiple advantages. The appropriate gene transduction of hUC-MSC before transplantation is a promising procedure for cell therapy. Fibroblast growth factor-20 (FGF-20) has been shown to protect dopaminergic neurons against a range of toxic insults in vitro. In this study, the hUC-MSCs were gene transduced with FGF-20, and then we transplanted them into the PD mice model. The results showed that MSC-FGF-20 treatment obviously improved the behavior of PD, accompanied by the increase of tyrosine carboxylase- (TH-) positive cell and dopamine (DA). Furtherly, immunohistochemistry disclosed that MSC-FGF-20 obviously promoted the degradation of nuclear factor-κB (NF-κB), a transcription factor that controls genes encoding proinflammatory cytokines, highly expressed in the nigrostriatal dopaminergic regions in PD patients. Therefore, MSC-FGF-20 has a potential for improving PD, closely related to the degradation of NF-κB. PMID:27274736

  13. X-ray Diffraction Studies of the Thick Filament in Permeabilized Myocardium from Rabbit

    SciTech Connect

    Xu,S.; Martyn, D.; Zaman, J.; Yu, L.

    2007-01-01

    Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25{sup o}C to 5{sup o}C at 200 mM and 50 mM ionic strengths ({mu}), respectively. Effects of temperature and {mu} on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I{sub 1,1}/I{sub 1,0}, and the spacing of the filament lattice are similar in both muscles. At 25{sup o}C, particularly at {mu} = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I{sub 1,1}/I{sub 1,0}. Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P{sub i}]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P{sub i} state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I{sub 1,1}/I{sub 1,0} ratio tends to be higher, and the lattice spacing D{sub 10}, larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.

  14. X-ray Diffraction Studies of the Thick Filament in Permeabilized Myocardium from Rabbit

    SciTech Connect

    Xu,S.; Martyn, D.; Zaman, J.; Yu, L.

    2006-01-01

    Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25{sup o}C to 5{sup o}C at 200 mM and 50 mM ionic strengths ({mu}), respectively. Effects of temperature and {mu} on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I{sub 1,1}/I{sub 1,0}, and the spacing of the filament lattice are similar in both muscles. At 25{sup o}C, particularly at {mu} = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I{sub 1,1}/I{sub 1,0}. Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P{sub i}]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P{sub i} state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I{sub 1,1}/I{sub 1,0} ratio tends to be higher, and the lattice spacing D{sub 10}, larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.

  15. Comparison of the biological effectiveness of 45 MeV C-ions and {gamma}-rays in inducing early and late effects in normal human primary fibroblasts

    SciTech Connect

    Fratini, E.; Balduzzi, M.; Antonelli, F.; Sorrentino, E.; Esposito, G.; Cuttone, G.; Romano, F.; Dini, V.; Simone, G.; Campa, A.; Tabocchini, M. A.; Belli, M.

    2013-07-18

    Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study compares early and late effects induced in AG01522 normal human primary fibroblasts by {gamma}-rays and C-ions having E {approx} 45 MeV/u at the cell entrance, corresponding to LET (in water) {approx} 49 keV/{mu}m. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN); DNA damage, in terms of DSB induction and repair, as measured by the H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and induction of lethal mutations, with RBEs of about 1.3 and 1.6 respectively, indicating that the presence of genomic instability is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for {gamma}-rays than for C-ions. However, in the first 12 h the fraction of residual {gamma}-H2AX foci was higher for C-ions irradiated cells, indicating a lower removal rate, possibly related to multiple/more complex damage along the particle track, with respect to the sparse lesions produced by {gamma}-rays. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to {gamma}-rays, likely

  16. Comparison of the biological effectiveness of 45 MeV C-ions and γ-rays in inducing early and late effects in normal human primary fibroblasts

    NASA Astrophysics Data System (ADS)

    Fratini, E.; Balduzzi, M.; Antonelli, F.; Sorrentino, E.; Esposito, G.; Cuttone, G.; Romano, F.; Dini, V.; Simone, G.; Belli, M.; Campa, A.; Tabocchini, M. A.

    2013-07-01

    Investigation of the mechanisms underlying the biological effects induced by densely ionizing radiation has relevant implications in both radiation protection and therapy. In particular, the possible advantages of hadrontherapy with respect to conventional radiotherapy in terms of high conformal tumor treatment and sparing of healthy tissues are well known. Further improvements are limited by lack of radiobiological knowledge, particularly about the specific cellular response to the damage induced by particles of potential interest for tumor treatment. This study compares early and late effects induced in AG01522 normal human primary fibroblasts by γ-rays and C-ions having E ˜ 45 MeV/u at the cell entrance, corresponding to LET (in water) ˜ 49 keV/μm. Different end points have been investigated, namely: cell killing and lethal mutation, evaluated as early and delayed reproductive cell death, respectively; chromosome damage, as measured by micronuclei induction (MN); DNA damage, in terms of DSB induction and repair, as measured by the H2AX phosphorylation/dephosphorylation kinetics. Linear dose-response relationships were found for cell killing and induction of lethal mutations, with RBEs of about 1.3 and 1.6 respectively, indicating that the presence of genomic instability is greater in the progeny of C-ions irradiated cells. H2AX phosphorylation/dephosphorylation kinetics have shown a maximum foci number at 30 min after irradiation, higher for γ-rays than for C-ions. However, in the first 12 h the fraction of residual γ-H2AX foci was higher for C-ions irradiated cells, indicating a lower removal rate, possibly related to multiple/more complex damage along the particle track, with respect to the sparse lesions produced by γ-rays. MN induction, observed after 72 h from irradiation, was also greater for C-ions. Overall, these data indicate a more severe DNA damage induced by 45 MeV/u C-ions with respect to γ-rays, likely responsible of an increased cellular

  17. ATP differentially upregulates fibroblast growth factor 2 and transforming growth factor α in neonatal and adult mice: effect on neuroproliferation.

    PubMed

    Jia, C; Cussen, A R; Hegg, C C

    2011-03-17

    Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. PMID:21187124

  18. HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors

    PubMed Central

    Taylor, Sarah E.; Mao, Xianqing; Wilkinson, Mark C.

    2015-01-01

    The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins. PMID:26137434

  19. Paracrine effect of GTP cyclohydrolase and angiopoietin-1 interaction in stromal fibroblasts on tumor Tie2 activation and breast cancer growth.

    PubMed

    Chen, Liye; Zeng, Xin; Kleibeuker, Esther; Buffa, Francesca; Barberis, Alessandro; Leek, Russell D; Roxanis, Ioannis; Zhang, Wei; Worth, Andrew; Beech, John S; Harris, Adrian L; Cai, Shijie

    2016-02-23

    Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer. PMID:26814432

  20. Paracrine effect of GTP cyclohydrolase and angiopoietin-1 interaction in stromal fibroblasts on tumor Tie2 activation and breast cancer growth

    PubMed Central

    Kleibeuker, Esther; Buffa, Francesca; Barberis, Alessandro; Leek, Russell D.; Roxanis, Ioannis; Zhang, Wei; Worth, Andrew; Beech, John S.; Harris, Adrian L.; Cai, Shijie

    2016-01-01

    Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer. PMID:26814432

  1. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

    PubMed Central

    2013-01-01

    Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

  2. Matrix metalloproteinase (MMP)-19-deficient fibroblasts display a profibrotic phenotype.

    PubMed

    Jara, Paul; Calyeca, Jazmin; Romero, Yair; Plácido, Luis; Yu, Guoying; Kaminski, Naftali; Maldonado, Vilma; Cisneros, José; Selman, Moisés; Pardo, Annie

    2015-03-15

    Idiopathic pulmonary fibrosis (IPF) is a progressive and usually lethal interstitial lung disease of unknown etiology characterized by aberrant activation of epithelial cells that induce the migration, proliferation and activation of fibroblasts. The resulting distinctive fibroblastic/myofibroblastic foci are responsible for the excessive extracellular matrix (ECM) production and abnormal lung remodeling. We have recently found that matrix metalloproteinase 19 (MMP-19)-deficient (Mmp19-/-) mice develop an exaggerated bleomycin-induced lung fibrosis, but the mechanisms are unclear. In this study, we explored the effect of MMP-19 deficiency on fibroblast gene expression and cell behavior. Microarray analysis of Mmp19-/- lung fibroblasts revealed the dysregulation of several profibrotic pathways, including ECM formation, migration, proliferation, and autophagy. Functional studies confirmed these findings. Compared with wild-type mice, Mmp19-/- lung fibroblasts showed increased α1 (I) collagen gene and collagen protein production at baseline and after transforming growth factor-β treatment and increased smooth muscle-α actin expression (P < 0.05). Likewise, Mmp19-deficient lung fibroblasts showed a significant increase in proliferation (P < 0.01) and in transmigration and locomotion over Boyden chambers coated with type I collagen or with Matrigel (P < 0.05). These findings suggest that, in lung fibroblasts, MMP-19 has strong regulatory effects on the synthesis of key ECM components, on fibroblast to myofibroblast differentiation, and in migration and proliferation. PMID:25575513

  3. Mesenchymal stem cells induce dermal fibroblast responses to injury

    SciTech Connect

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  4. Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

    PubMed Central

    O'Sullivan, A J; Brown, A M; Freeman, H N; Gomperts, B D

    1996-01-01

    Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown. Images PMID:8868468

  5. Monitoring the permeabilization of a single cell in a microfluidic device, through the estimation of its dielectric properties based on combined dielectrophoresis and electrorotation in situ experiments.

    PubMed

    Trainito, Claudia Irene; Français, Olivier; Le Pioufle, Bruno

    2015-05-01

    The electric field is commonly used in microdevices to handle, treat, or monitor living cells for various biological or biomedical applications (cells electrofusion, gene electrotransfer, drugs injection, cell sorting, …). Dielectrophoresis (DEP) forces, using stationary waves (conventional DEP) or traveling waves, are widely used for the cell handling or sorting. Electrorotation, which is induced by a rotating electrical field, is used for the determination of cell dielectric parameters. The application of pulsed electric field (PEF) results in the cell membrane permeabilization that might allow the transfer of various molecules in the cytoplasm. In this paper, we propose a method to monitor in situ the level of electropermeabilization induced by PEF application on a single cell, by combining the dielectrophoresis force and the electrorotation torque within a microfluidic device. The method was experimented on two different cell lines (human leukemic T-cell lymphoblast and murine melanoma cell): a single cell is captured by dielectrophoresis while its dielectric properties (both permittivity and conductivity of cytoplasm and membrane) are estimated thanks to a rotating electric field, which is applied simultaneously. The permeabilization effect of PEF, applied to the single cell trapped in such conditions in the biodevice, could be monitored by the estimation of its dielectric properties before and after pulse application. PMID:25641658

  6. Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex.

    PubMed

    Firsov, Alexander M; Kotova, Elena A; Korepanova, Evgeniya A; Osipov, Anatoly N; Antonenko, Yuri N

    2015-03-01

    Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation. PMID:25485477

  7. Pore size assessment during corneal endothelial cells permeabilization by femtosecond laser activated carbon nanoparticles

    NASA Astrophysics Data System (ADS)

    Jumelle, C.; Mauclair, C.; Houzet, J.; Bernard, A.; He, Z.; Piselli, S.; Perrache, C.; Egaud, G.; Baubeau, E.; Gain, P.; Thuret, G.

    2015-07-01

    Corneal therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells (HCECs) viability, but the difficulty is transport across cell membrane. A new delivery method published recently consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles (CNPs) and femtosecond laser (FsL). The aim of this work is to investigate the size of pores formed at cell membrane by this technique. To induce cell permeabilization, HCECs were put in contact with CNPs and irradiated with a 500 μm diameter Ti:Sa FsL focalized spot. Four sizes of marker molecules were delivered into HCECs to investigate pore sizes: calcein (1.2 nm), FITC-Dextran 4kDa (2.8 nm) and FITC-Dextran 70kDa (12 nm) and FITC-Dextran 2MDa (50 nm). Delivery of each molecule was assessed by flow cytometry, a technique able to measure their presence into cells. We showed that the delivery rate was dependent of their size. Calcein was delivered in 56.1±8.2% of HCECs, FITC-Dextran 4kDa in 42.2±3.5%, FITC-Dextran 70 kDa in 21.5±2.7% and finally FITC-Dextran 2MDa in 12.9±2.0%. It means that a large number of pores in the size ranging from 1.2 to 2.8 nm were formed. However, 12 nm and larger pores were almost half more infrequent. Pore sizes formed at cell membrane by the technique of cell permeabilization by FsL activated CNPs was investigated. The results indicated that the pore sizes are large enough for the efficient delivery of small, medium and big therapeutics molecules on HCECs by this technique.

  8. The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

    PubMed

    Bittencourt, L S; Machado, D C; Machado, M M; Dos Santos, G F F; Algarve, T D; Marinowic, D R; Ribeiro, E E; Soares, F A A; Barbisan, F; Athayde, M L; Cruz, I B M

    2013-03-01

    The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 μM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels. PMID:23220610

  9. Protective and restorative effects of a Commiphora mukul gum resin and triheptanoin preparation on the CCL-110 skin fibroblast cell line.

    PubMed

    Ramachandran, C; Quirin, K-W; Resek, A; Melnick, S J

    2012-04-01

    Coenzyme Q10 (CoQ10) is a major ingredient in skin care products because of its anti-wrinkle effects, although it has some side effects especially at higher amounts. In this study, we compare the anti-wrinkle related properties of CoQ10 and a proprietary Commiphora mukul gum resin (guggul) and triheptanoin preparation (GU-TC7). GU-TC7 is prepared with a supercritical CO₂-co-solvent extraction with ethanol, standardized to 2% guggulsterones and triheptanoin, a triglyceride composed of three 7-carbon fatty acids. Treatment of CCL-110 skin fibroblasts with GU-TC7 demonstrates a mild proliferative effect compared to CoQ10 and increased type I collagen synthesis. Additionally, GU-TC7 inhibited matrix metalloproteinase-1 (MMP-1) expression in a dose-dependent manner at 20-100 μg mL⁻¹ and inhibited human elastase expression by more than 50% as compared to no elastase inhibition with CoQ10 treatment. These results suggest that GU-TC7 possesses properties that are applicable to the treatment of wrinkles and may be considered for its further evaluation in skin care products. PMID:22084831

  10. Dual blockade of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) exhibits potent anti-angiogenic effects.

    PubMed

    Li, Dong; Xie, Kun; Zhang, Longzhen; Yao, Xuejing; Li, Hongwen; Xu, Qiaoyu; Wang, Xin; Jiang, Jing; Fang, Jianmin

    2016-07-28

    Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF or FGF-2) are potent pro-angiogenic factors and play a critical role in cancer development and progression. Clinical anti-VEGF therapy trials had a major challenge due to upregulated expression of other pro-angiogenic factor, like FGF-2. This study developed a novel chimeric decoy receptor VF-Trap fusion protein to simultaneously block activity of both VEGF and FGF pathways in order to achieve an additive or synergistic anti-tumor effect. Our in vitro data showed that VF-Trap potently blocked proliferation and migration of both VEGF- and FGF-2-induced vascular endothelial cells. In animal models, treatment of xenograft tumors with VF-Trap resulted in significant inhibition of tumor growth compared to blockage of the single molecule, like VEGF or FGF blocker. In addition, VF-Trap was also more potent in inhibition of ocular angiogenesis in a mouse oxygen-induced retinopathy (OIR) model. These data demonstrated the potent anti-angiogenic effects of this novel VF-Trap fusion protein on blockage of VEGF and FGF-2 activity in vitro and in animal models. Further study will assess its effects in clinic as a therapeutic agent for angiogenesis-related disorders, such as cancer and ocular vascular diseases. PMID:27130666

  11. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    SciTech Connect

    Nobe, Koji; Nobe, Hiromi; Yoshida, Hiroko; Kolodney, Michael S.; Paul, Richard J.; Honda, Kazuo

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  12. Effects of Ginger Phenylpropanoids and Quercetin on Nrf2-ARE Pathway in Human BJ Fibroblasts and HaCaT Keratinocytes

    PubMed Central

    Schadich, Ermin; Hlaváč, Jan; Volná, Tereza; Varanasi, Lakshman; Hajdúch, Marián; Džubák, Petr

    2016-01-01

    Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells. PMID:26942188

  13. Effects of the antimicrobial peptide cathelicidin (LL-37) on immortalized gingival fibroblasts infected with Porphyromonas gingivalis and irradiated with 625-nm LED light.

    PubMed

    Kim, JiSun; Kim, SangWoo; Lim, WonBong; Choi, HongRan; Kim, OkJoon

    2015-11-01

    Porphyromonas gingivalis causes chronic inflammatory diseases (periodontal diseases) that destroy the periodontal ligament and alveolar bone. Antimicrobial peptides are crucial components of the host defense response required to maintain cellular homeostasis during microbial invasion. Because light-emitting diode (LED) irradiation influences the host defense response against bacterial infections, we investigated its effect on immortalized gingival fibroblasts (IGFs) infected with P. gingivalis. IGFs were incubated with P. gingivalis following LED irradiation at 425, 525, and 625 nm. The dark 1 group comprised noninfected, nonirradiated IGFs, and the dark 2 group comprised nonirradiated IGFs infected with P. gingivalis. These groups served as controls. Infected cells and controls were assayed for reactive oxygen species (ROS) and were subjected to RT-PCR and Western blotting analyses to determine the levels of expression of antimicrobial peptides. LED irradiation enhanced the bactericidal effects of the antimicrobial peptide LL-37 in cells infected with P. gingivalis. Irradiation at 625 nm decreased inflammatory responses involving the release of prostaglandin E2 induced by ROS in P. gingivalis-infected IGFs. LED irradiation at 625 nm induces an anti-inflammatory response that elicits the production of antimicrobial peptides, providing an efficacious method of treatment for periodontal diseases. PMID:25543295

  14. Lack of effects on key cellular parameters of MRC-5 human lung fibroblasts exposed to 370 mT static magnetic field.

    PubMed

    Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; d'Angelo, Raffaele; Zeni, Olga

    2016-01-01

    The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components. PMID:26762783

  15. Fibroblast growth factor 18 increases the trophic effects of bone marrow mesenchymal stem cells on chondrocytes isolated from late stage osteoarthritic patients.

    PubMed

    Zhang, Zhenyu; Wang, Yan; Li, Mingchao; Li, Jiaping; Wu, Jian

    2014-01-01

    Coculture of mesenchymal stem cells with chondrocytes increases production of cartilaginous matrix. Chondrocytes isolated from late stage osteoarthritic patients usually lost their phenotype of producing cartilaginous matrix. Fibroblast growth factor 18 is believed to redifferentiate OA chondrocyte into functionally active chondrocytes. The aim of this study is to investigate the supportive effects of MSCs on OA chondrocytes and test if FGF18 could enhance the responsiveness of OA chondrocytes to the support of MSCs in a coculture system. Both pellet and transwell co-cultures were used. GAG quantification, hydroxyproline assay, and qPCR were performed. An ectopic models of cartilage formation was also applied. Our data indicated that, in pellets coculture of MSCs and OA chondrocytes, matrix production was increased in the presence of FGF18, comparing to the monoculture of chondrocytes. Results from transwell coculture study showed that expression of matrix producing genes in OA chondrocytes increased when cocultured with MSCs with FGF18 in culture medium, while hypertrophic genes were not changed by coculture. Finally, coimplantation of MSCs with OA chondrocytes produces more matrix than chondrocytes only. In conclusion, FGF18 can restore the responsiveness of OA chondrocytes to the trophic effects of MSCs. Coimplantation of MSCs and OA chondrocytes treated with FGF18 may be a good alternative cell source for regenerating cartilage tissue that is degraded during OA pathological changes. PMID:25544847

  16. The Effect of Walterinnesia aegyptia Venom Proteins on TCA Cycle Activity and Mitochondrial NAD+-Redox State in Cultured Human Fibroblasts

    PubMed Central

    Ghneim, Hazem K.; Al-Sheikh, Yazeed A.; Aboul-Soud, Mourad A. M.

    2015-01-01

    Fibroblast cultures were used to study the effects of crude Walterinnesia aegyptia venom and its F1–F7 protein fractions on TCA cycle enzyme activities and mitochondrial NAD-redox state. Confluent cells were incubated with 10 μg of venom proteins for 4 hours at 37°C. The activities of all studied TCA enzymes and the non-TCA mitochondrial NADP+-dependent isocitrate dehydrogenase underwent significant reductions of similar magnitude (50–60% of control activity) upon incubation of cells with the crude venom and fractions F4, F5, and F7 and 60–70% for fractions F3 and F6. In addition, the crude and fractions F3–F7 venom proteins caused a drop in mitochondrial NAD+ and NADP+ levels equivalent to around 25% of control values. Whereas the crude and fractions F4, F5, and F7 venom proteins caused similar magnitude drops in NADH and NADPH (around 55% of control levels), fractions F3 and F6 caused a more drastic drop (60–70% of control levels) of both reduced coenzymes. Results indicate that the effects of venom proteins could be directed at the mitochondrial level and/or the rates of NAD+ and NADP+ biosynthesis. PMID:25705684

  17. The effect of Walterinnesia aegyptia venom proteins on TCA cycle activity and mitochondrial NAD(+)-redox state in cultured human fibroblasts.

    PubMed

    Ghneim, Hazem K; Al-Sheikh, Yazeed A; Aboul-Soud, Mourad A M

    2015-01-01

    Fibroblast cultures were used to study the effects of crude Walterinnesia aegyptia venom and its F1-F7 protein fractions on TCA cycle enzyme activities and mitochondrial NAD-redox state. Confluent cells were incubated with 10 μg of venom proteins for 4 hours at 37°C. The activities of all studied TCA enzymes and the non-TCA mitochondrial NADP(+)-dependent isocitrate dehydrogenase underwent significant reductions of similar magnitude (50-60% of control activity) upon incubation of cells with the crude venom and fractions F4, F5, and F7 and 60-70% for fractions F3 and F6. In addition, the crude and fractions F3-F7 venom proteins caused a drop in mitochondrial NAD(+) and NADP(+) levels equivalent to around 25% of control values. Whereas the crude and fractions F4, F5, and F7 venom proteins caused similar magnitude drops in NADH and NADPH (around 55% of control levels), fractions F3 and F6 caused a more drastic drop (60-70% of control levels) of both reduced coenzymes. Results indicate that the effects of venom proteins could be directed at the mitochondrial level and/or the rates of NAD(+) and NADP(+) biosynthesis. PMID:25705684

  18. Non-Target Effect for Chromosome Aberrations in Human Lymphocytes and Fibroblasts After Exposure to Very Low Doses of High LET Radiation

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, Kerry A.; Cucinotta, F. A.

    2011-01-01

    The relationship between biological effects and low doses of absorbed radiation is still uncertain, especially for high LET radiation exposure. Estimates of risks from low-dose and low-dose-rates are often extrapolated using data from Japanese atomic bomb survivor with either linear or linear quadratic models of fit. In this study, chromosome aberrations were measured in human peripheral blood lymphocytes and normal skin fibroblasts cells after exposure to very low dose (.01 - 0.2 Gy) of 170 MeV/u Si-28-ions or 600 MeV/u Fe-56-ions. Chromosomes were analyzed using the whole chromosome fluorescence in situ hybridization (FISH) technique during the first cell division after irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). The curves for doses above 0.1 Gy were more than one ion traverses a cell showed linear dose responses. However, for doses less than 0.1 Gy, Si-28-ions showed no dose response, suggesting a non-targeted effect when less than one ion traversal occurs. Additional findings for Fe-56 will be discussed.

  19. Lack of effects on key cellular parameters of MRC-5 human lung fibroblasts exposed to 370 mT static magnetic field

    PubMed Central

    Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; d’Angelo, Raffaele; Zeni, Olga

    2016-01-01

    The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components. PMID:26762783

  20. Developmental regulation of the effects of fibroblast growth factor-2 and 1-octanol on neuronogenesis: implications for a hypothesis relating to mitogen-antimitogen opposition

    NASA Technical Reports Server (NTRS)

    Goto, T.; Takahashi, T.; Miyama, S.; Nowakowski, R. S.; Bhide, P. G.; Caviness, V. S. Jr

    2002-01-01

    Neocortical neurons arise from a pseudostratified ventricular epithelium (PVE) that lies within the ventricular zone (VZ) at the margins of the embryonic cerebral ventricles. We examined the effects of fibroblast growth factor-2 (FGF-2) and 1-octanol on cell output behavior of the PVE in explants of the embryonic mouse cerebral wall. FGF-2 is mitogenic and 1-octanol antimitogenic in the PVE. Whereas all postmitotic cells migrate out of the VZ in vivo, in the explants some postmitotic cells remain within the VZ. We refer to these cells as the indeterminate or I fraction, because they neither exit from the VZ nor reenter S phase as part of the proliferative (P) fraction. They are considered to be either in an extremely prolonged G(1) phase, unable to pass the G(1)/S transition, or in the G(0) state. The I fate choice is modulated by both FGF-2 and 1-octanol. FGF-2 decreased the I fraction and increased the P fraction. In contrast, 1-octanol increased the I fraction and nearly eliminated the P fraction. The effects of FGF-2 and 1-octanol were developmentally regulated, in that they were observed in the developmentally advanced lateral region of the cerebral wall but not in the medial region. Copyright 2002 Wiley-Liss, Inc.

  1. Fractal morphology of Beta vulgaris L. cell suspension culture permeabilized with Triton X-100®

    NASA Astrophysics Data System (ADS)

    Arenas-Ocampo, M.; Alamilla-Beltrán, L.; Vanegas-Espinoza, P.; Camacho-Díaz, B.; Campos-Mendiola, R.; Gutiérrez-López, G.; Jiménez-Aparicio, A.

    2012-02-01

    In this work, morphology of Beta vulgaris L. cells permeabilized with 0.7mM of Triton X-100® was evaluated using digital image processing and concepts of fractal dimension (perimeter- area relations). Important morphometric changes were found when the contact-time with chemical agent was increased. The size of cells decreased, the cells lost the roundness and their shape was more sinuous; this behaviour was a result of a probable shrinkage caused by the excess of exposure with the permeabilization agent. Morphology of B. vulgaris cells after permeabilization, exhibited a fractal nature since the slope of the ratio of the logarithm of the perimeter vs logarithm of the area was higher than unit. Fractal geometry of the cell morphology was affected as a result of the exposure to Triton X-100®. Those changes can be attributed to the loss of turgor and structure of the cell wall.

  2. Fluctuation of the CaS -sequestering activity of permeabilized sea urchin embryos during the cell cycle

    SciTech Connect

    Suprynowicz, F.A.; Mazia, D.

    1985-04-01

    The authors have followed the sequestration of CaS by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, UVCa, after filtration of aliquots on Millipore filters. The permeabilized cells sequester CaS at a constant rate for at least 20 min. The CaS -sequestering activities of embryos that are permeabilized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.

  3. Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells¶

    PubMed Central

    Jang, Young P.; Zhou, Jilin; Nakanishi, Koji; Sparrow, Janet R.

    2005-01-01

    The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon–carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. PMID:15745429

  4. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    PubMed

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. PMID:25849458

  5. IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions

    PubMed Central

    Kanekura, Kohsuke; Ma, Xiucui; Murphy, John T.; Zhu, Lihua J.; Diwan, Abhinav; Urano, Fumihiko

    2015-01-01

    The endoplasmic reticulum (ER) has emerged as a critical regulator of cell fate. IRE1 is a transmembrane protein with kinase and RNase activities that is localized to the ER and that promotes resistance to ER stress. Here we showed a mechanism by which IRE1 conferred protection against ER stress-mediated cell death. IRE1 signaling prevented ER membrane permeabilization mediated by Bax and Bak and cell death under ER stress conditions. Suppression of IRE1 signaling led to the accumulation of the BH3 domain-containing protein Bnip3, which in turn triggered the oligomerization of Bax and Bak in the ER membrane and ER membrane permeabilization. As a result, cells deficient in IRE1 were susceptible to leakage of ER contents in response to ER stress, which was associated with the accumulation of calcium in mitochondria, oxidative stress in the cytosol, and cell death. Our results reveal a role for IRE1 in preventing an initial step of cell death emanating from the ER and provide a potential target for treating diseases characterized by ER stress, including diabetes and Wolfram syndrome. PMID:26106220

  6. DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity.

    PubMed

    Benameur, Laila; Auffan, Mélanie; C