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Sample records for permeabilized fibroblasts effects

  1. Polyethyleneimine is an effective permeabilizer of gram-negative bacteria.

    PubMed

    Helander, I M; Alakomi, H L; Latva-Kala, K; Koski, P

    1997-10-01

    The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 micrograms ml-1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.

  2. Carnitine-acylcarnitine translocase deficiency with severe hypoglycemia and auriculo ventricular block. Translocase assay in permeabilized fibroblasts.

    PubMed Central

    Pande, S V; Brivet, M; Slama, A; Demaugre, F; Aufrant, C; Saudubray, J M

    1993-01-01

    Deficiency of the enzymes of mitochondrial fatty acid oxidation and related carnitine dependent steps have been shown to be one of the causes of the fasting-induced hypoketotic hypoglycemia. We describe here carnitine-acylcarnitine translocase deficiency in a neonate who died eight days after birth. The proband showed severe fasting-induced hypoketotic hypoglycemia, high plasma creatine kinase, heartbeat disorder, hypothermia, and hyperammonemia. The plasma-free carnitine on day three was only 3 microM, and 92% of the total carnitine (37 microM) was present as acylcarnitine. Treatments with intravenous glucose, carnitine, and medium-chain triglycerides had been tried without improvements. Measurements in fibroblasts confirmed deficient oxidation of palmitate and showed normal activities of the carnitine palmitoyltransferases I and II and of the three acyl-CoA dehydrogenases. A total deficiency of the carnitine-acyl-carnitine translocase was found in fibroblasts using the carnitine acetylation assay (1986. Biochem. J. 236:143-148). This assay has been further simplified by seeking conditions permitting application to permeabilized fibroblasts and lymphocytes. Images PMID:8450053

  3. 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

  4. Weakening Effect of Cell Permeabilizers on Gram-Negative Bacteria Causing Biodeterioration

    PubMed Central

    Alakomi, H.-L.; Paananen, A.; Suihko, M.-L.; Helander, I. M.; Saarela, M.

    2006-01-01

    Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products. PMID:16820461

  5. Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration.

    PubMed

    Alakomi, H-L; Paananen, A; Suihko, M-L; Helander, I M; Saarela, M

    2006-07-01

    Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

  6. 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.

  7. [Effect of permeabilization on sulfate reduction activity of Desulfovibrio vulgaris Hildenborough cells in the presence of different electron donors].

    PubMed

    Xu, Hui-Wei; Zhang, Xu; Li, Li-Ming; Zheng, Guang-Jie; Li, Guang-He

    2013-01-01

    The Desulfovibrio vulgaris Hildenborough (DvH) cells permeabilized with ethanol were used as biocatalysts to enhance hydrogenotrophic sulfate conversion. The effect of permeabilization extent of DvH cells on sulfate reduction was studied in the presence of different electron donors. When hydrogen was used as an electron donor, the highest level of sulfate reduction activity attained in cells treated with 10% ethanol (V/V), followed by 15% -ethanol treated cells. Furthermore, sulfate reduction activity markedly decreased when the ethanol concentration exceeded 15%. However, when lactate was used as the electron donor, the optimum ethanol concentration of the permeabilizing reagent was 20%, followed by 15% and 10%. Even when ethanol concentration reached 25%, DvH cells remained their partial activity with lactate. In a word, sulfate reduction activity of DvH cells responded differently in the presence of different donors. This was because the oxidation process of H2 and lactate occurred at different positions in DvH cells, and consequently intracellular electron transport pathway differed. To ensure the integrity of the electron transport chain between the donor and the accepter was a key factor for determining the permeabilization extent and for the application of cell permeabilization technology.

  8. 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

  9. Effect of polyethylenimine, a cell permeabilizer, on the photosensitized destruction of algae by methylene blue and nuclear fast red.

    PubMed

    McCullagh, Cathy; Robertson, Peter K J

    2006-01-01

    The present study reports the effect a cell permeabilizer, polyethylenimine (PEI) has on the photodynamic effect of methylene blue (MB) and nuclear fast red (NFR) in the presence of hydrogen peroxide (H2O2). The photosensitized destruction of the algae Chlorella vulgaris under irradiation with visible light is examined. The photodynamic effect was investigated under aerobic and anaerobic conditions. The presence of a permeabilizer during the photosensitized destruction of C. vulgaris does not enhance the activity of the MB, MB/H2O2 system or the NFR, NFR/H2O2 system under aerobic conditions. However under anaerobic conditions we have determined that when a cell permeabilizer was added to the MB/H202 system, the photosensitized destruction of C. vulgaris proceeded via a combination of Type I and Type II mechanisms. The presence of PEI enforces MB/H2O2 to be active toward the destruction of C. vulgaris whether oxygen is present or absent. Under aerobic and anaerobic conditions the activity of NFR was suppressed in the presence of PEI as a result of electrostatic interactions between the photosensitizer and the cell permeabilizer. The decrease in fluorescence recorded is indicative of destruction of the chlorophyll a pigment.

  10. 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.

  11. A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle

    SciTech Connect

    Sullivan, T.S.; Wells, J.N. )

    1991-03-11

    Phorbol esters can induce contraction of vascular smooth muscle and potentiate calcium-induced contractions of permeabilized smooth muscle strips. The authors have used a synthetic peptide inhibitor based on residues 19-31 of PKC (PKC-I) to determine the importance of PKC in the PDBu potentiation of calcium-induced contractions in permeabilized coronary artery smooth muscle. Although peptides similar to PKC-I have been shown to also inhibit MLCK in vitro, MLCK was presumably not inhibited in our system since 30 {mu}M PKC-I alone did not alter the calcium-induced contractions. However, the potentiation of these contractions by 1 {mu}M PDBu was reduced by about 50% in the presence of 10 {mu}M PKC-I, and the potentiation was completely abolished by 30 {mu}M PKC-I. These data indicate that, in this system, PKC is not involved in calcium-induced contractions but that activation of PKC may be the mechanism by which PDBu potentiates calcium-induced contractions in permeabilized coronary artery smooth muscle.

  12. 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.

  13. 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

  14. Effects of vitamin E supplementation on plasma membrane permeabilization and fluidization induced by chlorpromazine in the rat brain.

    PubMed

    Maruoka, Nobuyuki; Murata, Tetsuhito; Omata, Naoto; Takashima, Yasuhiro; Fujibayashi, Yasuhisa; Wada, Yuji

    2008-03-01

    Neurotransmitter receptors play a key role in most research on antipsychotic drugs, but little is known about the effects of these drugs on the plasma membrane in the central nervous system. Therefore, we investigated whether chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, affects the plasma membrane integrity in the rat brain, and if so, whether these membrane alterations can be prevented by dietary supplementation with vitamin E, which has been shown to be an antioxidant and also a membrane-stabilizer. Leakage of [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG)-6-phosphate from rat striatal slices and decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy were used as indexes for plasma membrane permeabilization and fluidization, respectively. CPZ induced leakage of [(18)F]FDG-6-phosphate from striatal slices, and the leakage was delayed in the vitamin E-supplemented group compared to that in the normal diet group. The decrease in plasma membrane anisotropy induced by CPZ was significantly attenuated by vitamin E supplementation. Chronic treatment with alpha-phenyl-N-tert-butyl nitrone, a free radical scavenger, had no effect on CPZ-induced plasma membrane permeabilization, and the treatment with CPZ did not induce lipid peroxidation. CPZ can reduce plasma membrane integrity in the brain, and this reduction can be prevented by vitamin E via its membrane-stabilizing properties, not via its antioxidant activity.

  15. Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus.

    PubMed

    Valcarcel, C A; Dalla Serra, M; Potrich, C; Bernhart, I; Tejuca, M; Martinez, D; Pazos, F; Lanio, M E; Menestrina, G

    2001-06-01

    Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory

  16. Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus.

    PubMed Central

    Valcarcel, C A; Dalla Serra, M; Potrich, C; Bernhart, I; Tejuca, M; Martinez, D; Pazos, F; Lanio, M E; Menestrina, G

    2001-01-01

    Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA >> PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small

  17. Effects of intermittent hypoxic training on amino and fatty acid oxidative combustion in human permeabilized muscle fibers.

    PubMed

    Roels, Belle; Thomas, Claire; Bentley, David J; Mercier, Jacques; Hayot, Maurice; Millet, Grégoire

    2007-01-01

    The effects of concurrent hypoxic/endurance training on mitochondrial respiration in permeabilized fibers in trained athletes were investigated. Eighteen endurance athletes were divided into two training groups: normoxic (Nor, n = 8) and hypoxic (H, n = 10). Three weeks (W1-W3) of endurance training (5 sessions of 1 h to 1 h and 30 min per week) were completed. All training sessions were performed under normoxic [160 Torr inspired Po(2) (Pi(O(2)))] or hypoxic conditions ( approximately 100 Torr Pi(O(2)), approximately 3,000 m) for Nor and H group, respectively, at the same relative intensity. Before and after the training period, an incremental test to exhaustion in normoxia was performed, muscle biopsy samples were taken from the vastus lateralis, and mitochondrial respiration in permeabilized fibers was measured. Peak power output (PPO) increased by 7.2% and 6.6% (P < 0.05) for Nor and H, respectively, whereas maximal O(2) uptake (Vo(2 max)) remained unchanged: 58.1 +/- 0.8 vs. 61.0 +/- 1.2 ml.kg(-1).min(-1) and 58.5 +/- 0.7 vs. 58.3 +/- 0.6 ml.kg(-1).min(-1) for Nor and H, respectively, between pretraining (W0) and posttraining (W4). Maximal ADP-stimulated mitochondrial respiration significantly increased for glutamate + malate (6.27 +/- 0.37 vs. 8.51 +/- 0.33 mumol O(2).min(-1).g dry weight(-1)) and significantly decreased for palmitate + malate (3.88 +/- 0.23 vs. 2.77 +/- 0.08 mumol O(2).min(-1).g dry weight(-1)) in the H group. In contrast, no significant differences were found for the Nor group. The findings demonstrate that 1) a 3-wk training period increased the PPO at sea level without any changes in Vo(2 max), and 2) a 3-wk hypoxic exercise training seems to alter the intrinsic properties of mitochondrial function, i.e., substrate preference.

  18. Ceramides with a pentadecasphingosine chain and short acyls have strong permeabilization effects on skin and model lipid membranes.

    PubMed

    Školová, Barbora; Janůšová, Barbora; Vávrová, Kateřina

    2016-02-01

    The composition and organization of stratum corneum lipids play an essential role in skin barrier function. Ceramides represent essential components of this lipid matrix; however, the importance of the individual structural features in ceramides is not fully understood. To probe the structure-permeability relationships in ceramides, we prepared analogs of N-lignoceroylsphingosine with shortened sphingosine (15 and 12 carbons) and acyl chains (2, 4 and 6 carbons) and studied their behavior in skin and in model lipid membranes. Ceramide analogs with pentadecasphingosine (15C) chains were more barrier-perturbing than 12C- and 18C-sphingosine ceramides; the greatest effects were found with 4 to 6C acyls (up to 15 times higher skin permeability compared to an untreated control and up to 79 times higher permeability of model stratum corneum lipid membranes compared to native very long-chain ceramides). Infrared spectroscopy using deuterated lipids and X-ray powder diffraction showed surprisingly similar behavior of the short ceramide membranes in terms of lipid chain order and packing, phase transitions and domain formation. The high- and low-permeability membranes differed in their amide I band shape and lamellar organization. These skin and membrane permeabilization properties of some short ceramides may be explored, for example, for the rational design of permeation enhancers for transdermal drug delivery.

  19. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  20. 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.

  1. Fatty acid effects on fibroblast cholesterol synthesis

    SciTech Connect

    Shireman, R.B.; Muth, J.; Lopez, C.

    1987-05-01

    Two cell lines of normal (CRL 1475, GM5565) and of familial hypercholesterolemia (FH) (CM 486,488) fibroblasts were preincubated with medium containing the growth factor ITS, 2.5 mg/ml fatty acid-free BSA, or 35.2 ..mu..mol/ml of these fatty acids complexed with 2.5 mg BSA/ml: stearic (18:0), caprylic (8:0), oleic (18:1;9), linoleic (18:2;9,12), linolenic (18:3;9,12,15), docosahexaenoic (22:6;4,7,10,13,16,19)(DHA) or eicosapentaenoic (20:5;5,8,11,14,17)(EPA). After 20 h, cells were incubated for 2 h with 0.2 ..mu..Ci (/sup 14/C)acetate/ml. Cells were hydrolyzed; an aliquot was quantitated for radioactivity and protein. After saponification and extraction with hexane, radioactivity in the aqueous and organic phases was determined. The FH cells always incorporated 30-90% more acetate/mg protein than normal cells but the pattern of the fatty acid effects was similar in both types. When the values were normalized to 1 for the BSA-only group, cells with ITS had the greatest (/sup 14/C)acetate incorporation (1.45) followed by the caprylic group (1.14). Cells incubated with 18:3, 20:6 or 22:6 incorporated about the same amount as BSA-only. Those preincubated with 18:2, 18:1, 18:0 showed the least acetate incorporation (0.87, 0.59 and 0.52, respectively). The percentage of total /sup 14/C counts which extracted into hexane was much greater in FH cells; however, these values varied with the fatty acid, e.g., 1.31(18:0) and 0.84(8:0) relative to 1(BSA).

  2. P2X7 receptor-pannexin 1 hemichannel association: effect of extracellular calcium on membrane permeabilization.

    PubMed

    Poornima, V; Madhupriya, M; Kootar, S; Sujatha, G; Kumar, Arvind; Bera, Amal Kanti

    2012-03-01

    Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2', 3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X(7)R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X(7)R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X(7)R-panx1, triggered by the removal of [Ca(2+)](o).

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

    PubMed

    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.

  4. Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

    PubMed

    Yoshimoto, A; Oki, T; Inui, T

    1978-10-04

    Exogenous addition of guanosine and adenosine 5'-(mono, di and tri) phosphate 3'-diphosphates (pppGpp, ppGpp, pGpp, pppApp, ppApp and pApp) stimulated the synthesis of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli. From the results obtained with ppGpp and pppApp, this effect appeared to be at a transcriptional level and depended greatly on the growth condition; the largest effect was observed in cells under shiftdown or grown on poor enrgy source. ppGpp and pppApp, unlike cyclic AMP, did not act to overcome the inhibition of enzyme induction by glucose, but in combination with cyclic AMP caused a synergistic stimulation effect. In the shiftdown cells, ppGpp and pppApp gave 30% or more stimulation effect on tryptophanase induction while cyclic AMP did not stimulate induction. There was therefore a pronounced difference between cyclic AMP and ppGpp or pppApp in stimulatory function.

  5. 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.

  6. Differential permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes as revealed by proteomics analysis of proteins released from mitochondria.

    PubMed

    Yamada, Akiko; Yamamoto, Takenori; Yamazaki, Naoshi; Yamashita, Kikuji; Kataoka, Masatoshi; Nagata, Toshihiko; Terada, Hiroshi; Shinohara, Yasuo

    2009-06-01

    It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224-5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.

  7. 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

  8. 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.

  9. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    SciTech Connect

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E. )

    1989-08-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.

  10. Tube extrusion from permeabilized giant vesicles

    NASA Astrophysics Data System (ADS)

    Borghi, N.; Kremer, S.; Askovic, V.; Brochard-Wyart, F.

    2006-08-01

    This letter reports the permeabilization effects of chemical additives on mechanical properties of Giant Unilamellar Vesicles (GUVs). We use a surfactant, Tween 20, inducing transient pores and a protein, Streptolysin O, inducing permanent pores in the membrane. Lipid tubes are extracted from GUVs anchored onto the tip of a micro-needle and submitted to hydrodynamic flows. On bare vesicles, tube extrusion is governed by the entropic elasticity of the membrane. The vesicle tension increases until it balances the flow velocity U and the tube reaches a stationary length. In permeabilized vesicles, the membrane tension is maintained at a constant value σp by the permeation of inner solution through nanometric pores. This allows extrusion of "infinite" tubes at constant velocity that never reach a stationary length. Tween-20 preliminary results suggest that σp strongly depends on surfactant concentration. For Streptolysin O, we have measured σp vs. U and found two regimes: a "high-porosity" regime for U > Up0 and a "low-porosity" regime for U < Up0, where Up0 is related to the number of pores on the vesicle surface.

  11. Effect of fibroblast-seeded artificial dermis on wound healing.

    PubMed

    Jang, Joon Chul; Choi, Rak-Jun; Han, Seung-Kyu; Jeong, Seong-Ho; Kim, Woo-Kyung

    2015-04-01

    In covering wounds, efforts should include use of the safest and least invasive methods with a goal of achieving optimal functional and cosmetic outcome. The recent development of advanced technology in wound healing has triggered the use of cells and/or biological dermis to improve wound healing conditions. The purpose of the study was to evaluate the effects of fibroblast-seeded artificial dermis on wound healing efficacy.Ten nude mice were used in this study. Four full-thickness 6-mm punch wounds were created on the dorsal surface of each mouse (total, 40 wounds). The wounds were randomly assigned to one of the following 4 treatments: topical application of Dulbecco phosphate-buffered saline (control), human fibroblasts (FB), artificial dermis (AD), and human fibroblast-seeded artificial dermis (AD with FB). On the 14th day after treatment, wound healing rate and wound contraction, which are the 2 main factors determining wound healing efficacy, were evaluated using a stereoimage optical topometer system, histomorphological analysis, and immunohistochemistry.The results of the stereoimage optical topometer system demonstrated that the FB group did not have significant influence on wound healing rate and wound contraction. The AD group showed reduced wound contraction, but wound healing was delayed. The AD with FB group showed decreased wound contraction without significantly delayed wound healing. Histomorphological analysis exhibited that more normal skin structure was regenerated in the AD with FB group. Immunohistochemistry demonstrated that the AD group and the AD with FB group produced less α-smooth muscle actin than the control group, but this was not shown in the FB group.Fibroblast-seeded artificial dermis may minimize wound contraction without significantly delaying wound healing in the treatment of skin and soft tissue defects.

  12. Effect of human serum albumin upon the permeabilizing activity of sticholysin II, a pore forming toxin from Stichodactyla heliantus.

    PubMed

    Celedón, Gloria; González, Gustavo; Gulppi, Felipe; Pazos, Fabiola; Lanio, María E; Alvarez, Carlos; Calderón, Cristian; Montecinos, Rodrigo; Lissi, Eduardo

    2013-12-01

    Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.

  13. Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

    PubMed Central

    Fu, Qiuli; Zhang, Lifang; Yin, Houfa; Jin, Xiuming; Tang, Qiaomei; Lyu, Danni

    2016-01-01

    Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L) on HPFs was lower than on HCFs (100 μmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R. PMID:27872516

  14. 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.

  15. Pressure effects on the growth of human scar fibroblasts.

    PubMed

    Chang, Liang-Wey; Deng, Win-Ping; Yeong, Eng-Kean; Wu, Ching-Yuan; Yeh, Shih-Wei

    2008-01-01

    Although pressure therapy is the mainstay of treatment for hypertrophic scars, its actual mechanism remains unknown. An in vitro study was designed to investigate the effects of positive pressure on the growth of human scar-derived fibroblasts through its transforming growth factor beta1 (TGF-beta1) secretion. A pneumatic pressure system connecting to a cell culture chamber was designed. Six-well cultured plates with fibroblasts implanted were treated with different pressure settings. Cells were treated with constant pressure 20 mm Hg above atmosphere pressure (group A n = 18) or with 40 mm Hg above atmosphere pressure (group B n = 18) daily for nine successive days. Cells without pressure were treated as the control study (group C n = 6). Each experimental group was divided into daily pressure applied at 24 hours (n = 6), 18 hours (n = 6), and 12 hours (n = 6). Cell counting was performed on the 2nd, 4th, 7th, 9th, 11th, and 14th day after implantation. On day 4, the concentration of transforming growth factor beta1 was measured, and cell doubling time was calculated. Compared with the control group, there was a significant decrease in cell count and the concentration in the 18-hour and 24-hour 20 mm Hg or 40 mm Hg pressure treated group. The cell doubling time was significantly increased in the 24-hour 20 mm Hg or 40 mm Hg pressure treated groups, and the 18-hour 40 mm Hg pressure treated group. (P < .05) Pressure inhibits the growth and activity of human scar fibroblasts, and a higher pressure application can shorten the daily application period. There should be an optimal pressure level corresponding to a daily application period to achieve the most effective results on pressure therapy for scars.

  16. Permeabilization of adhered cells using an inert gas jet.

    PubMed

    Cooper, Scott; Jonak, Paul; Chouinard-Pelletier, Guillaume; Coulombe, Sylvain; Jones, Elizabeth; Leask, Richard L

    2013-09-04

    Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes a mechanical method to temporally permeabilize adherent cells using an inert gas jet that can facilitate the transfer of normally non-permeable macromolecules into cells. We believe this technique works by imparting shear forces on the plasma membrane of adherent cells, resulting in the temporary formation of micropores. Once these pores are created, the cells are then permeable to genetic material and other biomolecules. The mechanical forces involved do run the risk of permanently damaging or detaching cells from their substrate. There is, therefore, a narrow range of inert gas dynamics where the technique is effective. An inert gas jet has proven efficient at permeabilizing various adherent cell lines including HeLa, HEK293 and human abdominal aortic endothelial cells. This protocol is appropriate for the permeabilization of adherent cells both in vitro and, as we have demonstrated, in vivo, showing it may be used for research and potentially in future clinical applications. It also has the advantage of permeabilizing cells in a spatially restrictive manner, which could prove to be a valuable research tool.

  17. Mitochondrial membrane permeabilization with nanosecond electric pulses.

    PubMed

    Vernier, P Thomas

    2011-01-01

    Ultra-short, high-field electric pulses permeabilize plasma and intracellular membranes. We report here nanosecond pulse-induced permeabilization of mitochondrial membranes in living cells. Using four independent methods based on fluorescent dyes--JC-1, rhodamine 123, tetramethyl rhodamine ethyl ester, and cobalt-quenched calcein--we show that as few as five, 4 ns, 10 MV/m pulses delivered at 1 kHz cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. The most likely interpretation of these results is a pulse-induced permeabilization of the inner mitochondrial membrane.

  18. 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.

  19. 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.

  20. 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.

  1. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

    PubMed Central

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

    2015-01-01

    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

  2. Constituents from the roots of Taraxacum platycarpum and their effect on proliferation of human skin fibroblasts.

    PubMed

    Warashina, Tsutomu; Umehara, Kaoru; Miyase, Toshio

    2012-01-01

    A MeOH extract from the roots of Taraxacum platycarpum has shown significant effects on the proliferation of normal human skin fibroblasts. Chemical analysis of the extract resulted in the isolation of 26 compounds, including eight new triterpenes, one new sesquiterpene glycoside, and seventeen known compounds. The structure of each new compound was established using NMR spectroscopy. Some triterpenes had a significant effect on the proliferation of normal human skin fibroblasts.

  3. 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.

  4. Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

    PubMed Central

    Wang, Yao-wen; Ren, Ji-hao; Xia, Kun; Wang, Shu-hui; Yin, Tuan-fang; Xie, Ding-hua; Li, Li-hua

    2012-01-01

    Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically. PMID

  5. 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

  6. Instability of spiral and scroll waves in the presence of a gradient in the fibroblast density: the effects of fibroblast-myocyte coupling

    NASA Astrophysics Data System (ADS)

    Zimik, Soling; Pandit, Rahul

    2016-12-01

    Fibroblast-myocyte coupling can modulate electrical-wave dynamics in cardiac tissue. In diseased hearts, the distribution of fibroblasts is heterogeneous, so there can be gradients in the fibroblast density (henceforth we call this GFD) especially from highly injured regions, like infarcted or ischemic zones, to less-wounded regions of the tissue. Fibrotic hearts are known to be prone to arrhythmias, so it is important to understand the effects of GFD in the formation and sustenance of arrhythmic re-entrant waves, like spiral or scroll waves. Therefore, we investigate the effects of GFD on the stability of spiral and scroll waves of electrical activation in a state-of-the-art mathematical model for cardiac tissue in which we also include fibroblasts. By introducing GFD in controlled ways, we show that spiral and scroll waves can be unstable in the presence of GFDs because of regions with varying spiral- or scroll-wave frequency ω, induced by the GFD. We examine the effects of the resting membrane potential of the fibroblast and the number of fibroblasts attached to the myocytes on the stability of these waves. Finally, we show that the presence of GFDs can lead to the formation of spiral waves at high-frequency pacing.

  7. Newcomers in the process of mitochondrial permeabilization.

    PubMed

    Lucken-Ardjomande, Safa; Martinou, Jean-Claude

    2005-02-01

    Under stress conditions, apoptogenic factors normally sequestered in the mitochondrial intermembrane space are released into the cytosol, caspases are activated and cells die by apoptosis. Although the precise mechanism that leads to the permeabilization of mitochondria is still unclear, the activation of multidomain pro-apoptotic proteins of the Bcl-2 family, such as Bax and Bak, is evidently crucial. Regulation of Bax and Bak by other members of the family has been known for a long time, but recent evidence suggests that additional unrelated proteins participate in the process, both as inhibitors and activators. The important rearrangements mitochondrial lipids undergo during apoptosis play a role in the permeabilization process and this role is probably more central than first envisioned.

  8. Reconstitution of Nuclear Import in Permeabilized Cells

    PubMed Central

    Cassany, Aurelia; Gerace, Larry

    2012-01-01

    The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin–cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin α, importin β, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes. PMID:18951186

  9. Reconstitution of nuclear import in permeabilized cells.

    PubMed

    Cassany, Aurelia; Gerace, Larry

    2009-01-01

    The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin-cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin alpha, importin beta, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.

  10. 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

  11. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts.

    PubMed

    Germeyer, Ariane; Sharkey, Andrew Mark; Prasadajudio, Mirari; Sherwin, Robert; Moffett, Ashley; Bieback, Karen; Clausmeyer, Susanne; Masters, Leanne; Popovici, Roxana Maria; Hess, Alexandra Petra; Strowitzki, Thomas; von Wolff, Michael

    2009-01-01

    The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. The dose-dependence biological effect of laser fluence on rabbit fibroblasts derived from urethral scar.

    PubMed

    Yang, Yong; Yu, Bo; Sun, Dongchong; Wu, Yuanyi; Xiao, Yi

    2015-04-01

    Two-micrometer laser vaporization resection has been used in clinic for years, but some patients received the treatment are still faced with excessive and abnormal wound repair which leads to the recurrent of urethral stricture eventually. Fibroblasts play a key role in the processes of "narrow-expansion/operation-restenosis" recurring problems. Here, we investigated the effect of laser fluence biomodulation on urethral scar fibroblasts as well as the underlying mechanism. Urethral scar fibroblasts were isolated and cultured, and laser irradiation (2 μm) was applied at different laser fluence or doses (0, 0.125, 0.5, 2, 8, 32 J/cm(2)) with a single exposure in 1 day. The effect of 2-μm laser irradiation on cell proliferation, viability, and expression of scar formation related genes were investigated. Two-micrometer laser irradiation with intermediate dose (8 J/cm(2)) promoted scar fibroblasts proliferation and reactive oxygen species (ROS) production, while higher doses of 32 J/cm(2) are suppressive as it decreased the survival rate, viability, and proliferation of fibroblasts. In addition, qRT-PCR and Western blotting results both proven that collagen type I, collagen IV, MMP9, and CTGF display significant increase, yet the TGF-β1 expression was severely reduced at intermediate dose (8 J/cm(2)) group when compared with the others groups. Our findings suggest the scar formation-related genes are sensitive to intermediate laser irradiation dose, the most in scar fibroblasts. We revealed the bioeffect and molecular mechanism of 2-μm laser irradiation on rabbit urethral scar fibroblasts. Our study provides new insights into the mechanisms which involved in the excessive and abnormal wound repair of 2-μm laser vaporization resection. These results could potentially contribute to further study on biological effects and application of 2-μm laser irradiation in urethral stricture therapy.

  17. Bnip3 Mediates Permeabilization of Mitochondria and Release of Cytochrome c via a Novel Mechanism

    PubMed Central

    Quinsay, Melissa N.; Lee, Youngil; Rikka, Shivaji; Sayen, M. Richard; Molkentin, Jeffery D.; Gottlieb, Roberta A.; Gustafsson, Åsa B.

    2010-01-01

    Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with loss of cardiac myocytes after a myocardial infarction. Previous studies have demonstrated that Bnip3 induces mitochondrial dysfunction, but the mechanisms involved in this process remain unknown. In this study, we demonstrate that Bnip3 induces permeabilization of the mitochondria via a novel mechanism that is different from other BH3-only proteins. We found that Bnip3 induced mitochondrial swelling and cytochrome c release in isolated heart mitochondria in vitro. Another BH3-only protein, tBid, also caused release of cytochrome c but failed to induce swelling of mitochondria. Swelling of mitochondria is a characteristic of mitochondrial permeability transition pore (mPTP) opening, but Bnip3-mediated mitochondrial swelling was insensitive to cyclosporine A, an inhibitor of the mPTP and independent of cyclophilin D (cypD), an essential component of the mPTP. Bnip3 also induced permeabilization of the mitochondrial membranes as evident by calcein release from the matrix in both wild type (WT) and cypD deficient mouse embryonic fibroblasts (MEFs). Moreover, Bnip3 induced mitochondrial matrix remodeling and large amplitude swelling of the inner membrane, which led to disassembly of OPA1 complexes and release from the mitochondria. Thus, these studies suggest that Bnip3 mediates mitochondrial permeabilization by a novel mechanism that is different from other BH3-only proteins. PMID:20025887

  18. Protective effect of kaempferol on LPS plus ATP-induced inflammatory response in cardiac fibroblasts.

    PubMed

    Tang, Xi-Lan; Liu, Jian-Xun; Dong, Wei; Li, Peng; Li, Lei; Hou, Jin-Cai; Zheng, Yong-Qiu; Lin, Cheng-Ren; Ren, Jun-Guo

    2015-02-01

    Inflammatory response is an important mechanism in the pathogenesis of cardiovascular diseases. Cardiac fibroblasts play a crucial role in cardiac inflammation and might become a potential therapeutic target in cardiovascular diseases. Kaempferol, a flavonoid commonly existing in many edible fruits, vegetables, and Chinese herbs, is well known to possess anti-inflammatory property and thus has a therapeutic potential for the treatment of inflammatory diseases. To date, the effect of kaempferol on cardiac fibroblasts inflammation is unknown. In this study, we investigated the anti-inflammatory effect of kaempferol on lipopolysaccharide (LPS) plus ATP-induced cardiac fibroblasts and explored the underlying mechanisms. Our results showed that kaempferol at concentrations of 12.5 and 25 μg/mL significantly suppressed the release of TNF-α, IL-1β, IL-6, and IL-18 and inhibited activation of NF-κB and Akt in LPS plus ATP-induced cardiac fibroblasts. These findings suggest that kaempferol attenuates cardiac fibroblast inflammation through suppression of activation of NF-κB and Akt.

  19. 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.

  20. A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation

    PubMed Central

    1991-01-01

    Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate

  1. Effects of whole cigarette smoke on human gingival fibroblast adhesion, growth, and migration.

    PubMed

    Semlali, Abdelhabib; Chakir, Jamila; Rouabhia, Mahmoud

    2011-01-01

    The aim of this study was to investigate the effects of a single exposure to whole cigarette smoke on human gingival fibroblast behavior. Normal oral mucosa fibroblasts were exposed once to whole cigarette smoke for 5, 15, or 30 min, and then were used to analyze cell adhesion, β1-integrin expression, cell growth and viability, cell capacity to contract collagen gel, and cell migration following wound infliction. Our findings showed that when gingival fibroblasts were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell adhesion, a decrease in the number of β1-integrin-positive cells, increased LDH activity in the target cells, and reduced growth. The smoke-exposed fibroblasts were also not able to contract collagen gel matrix and migrate following insult. Overall results demonstrate that a single exposure to whole cigarette smoke produced significant morphological and functional deregulation in gingival fibroblasts. This may explain the higher predisposition of tobacco users to oral infections and diseases such as cancer.

  2. 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.

  3. An investigation into the effect of amphiphilic siloxane oligomers on dermal fibroblasts.

    PubMed

    Farrugia, Brooke L; Keddie, Daniel J; George, Graeme A; Lynam, Emily C; Brook, Michael A; Upton, Zee; Dargaville, Tim R

    2012-07-01

    This study investigates the effect of well-defined poly(dimethylsiloxane)-poly(ethylene glycol) (PDMS-PEG) ABA linear block co-oligomers on the proliferation of human dermal fibroblasts. The co-oligomers assessed ranged in molecular weight (MW) from 1335 to 5208 Da and hydrophilic-lipophilic balance (HLB) from 5.9 to 16.6 by varying the number of both PDMS and PEG units. In general, it was found that co-oligomers of low MW or intermediate hydrophilicity significantly reduced fibroblast proliferation. A linear relationship between down-regulation of fibroblast proliferation, and the ratio HLB/MW was observed at concentrations of 0.1 and 1.0 wt % of the oligomers. This enabled the structures with highest efficiency to be determined. These results suggest the possible use of the PEG-PDMS-PEG block co-oligomers as an alternative to silicone gels for hypertrophic scar remediation.

  4. 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.

  5. Effect of roughness of zirconia and titanium on fibroblast adhesion.

    PubMed

    Takamori, Esther Rieko; Cruz, Renato; Gonçalvez, Fábio; Zanetti, Raquel Virgínia; Zanetti, Artemio; Granjeiro, José Mauro

    2008-04-01

    The aim of this study was to investigate the adhesion (4 and 24 h) and the morphology of fibroblast Balb/c 3T3 seeded onto polystyrene, partially stabilized (ZrO(2)Y(2)O(3)), stabilized zirconia ceramic (3YTZP), and pure titanium (Ti, grade 2). Initial cell adhesion (4 h) was greater (P < 0.05, analysis of variance and Tukey's Multiple Comparisons Test) onto ZrO(2)Y(2)O(3) and polystyrene than in Ti and 3YTZ. After 24 h, the number of adhered cells was similar between the biomaterials tested, but smaller than onto polystyrene (P < 0.05). Cells were more spread onto glass surface after 4 h, but after 24 h, the morphology and density of the cells were similar in all groups (SEM). Profilometry showed distinct Ra values for each material: glass coverslips and ZrO(2)Y(2)O(3) (0.09 microm), Ti (0.88 microm), and 3YTZP (30.93 microm). It was concluded that ZrO(2)Y(2)O(3) promoted the best initial adhesion, thus indicating that surfaces with Ra values smaller than 0.1 microm could be more favorable to initial adhesion.

  6. Effects of alkaline treatment for fibroblastic adhesion on titanium

    PubMed Central

    Cuellar-Flores, Miryam; Acosta-Torres, Laura Susana; Martínez-Alvarez, Omar; Sánchez-Trocino, Benjamin; de la Fuente-Hernández, Javier; Garcia-Garduño, Rigoberto; Garcia-Contreras, Rene

    2016-01-01

    Background: The surface energy of titanium (Ti) implants is very important when determining hydrophilicity or hydrophobicity, which is vital in osseointegration. The purpose of this study was to determine how Ti plates with an alkaline treatment (NaOH) affect the adhesion and proliferation of human periodontal ligament fibroblasts (HPLF). Materials and Methods: In vitro experimental study was carried out. Type 1 commercially pure Ti plates were analyzed with atomic force microscopy to evaluate surface roughness. The plates were treated ultrasonically with NaOH at 5 M (pH 13.7) for 45 s. HPLF previously established from periodontal tissue was inoculated on the treated Ti plates. The adhered and proliferated viable cell numbers were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method for 60 min and 24 h, respectively. The data were analyzed using Kruskal–Wallis tests and multiple comparisons of the Mann–Whitney U-test,P value was fixed at 0.05. Results: The mean roughness values equaled 0.04 μm with an almost flat surface and some grooves. The alkaline treatment of Ti plates caused significantly (P < 0.05) more pronounced HPLF adhesion and proliferation compared to untreated Ti plates. Conclusion: The treatment of Ti plates with NaOH enhances cell adhesion and the proliferation of HPLF cells. Clinically, the alkaline treatment of Ti-based implants could be an option to improve and accelerate osseointegration. PMID:28182066

  7. 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).…

  8. The effects of platelet gel-released supernatant on human fibroblasts.

    PubMed

    Giusti, Ilaria; Rughetti, Anna; D'Ascenzo, Sandra; Di Stefano, Gabriella; Nanni, Maria Rita; Millimaggi, Danilo; Dell'orso, Luigi; Dolo, Vincenza

    2013-01-01

    In recent years, interest in the topical use of platelet gel (PG) to stimulate wound healing has rapidly extended into various clinical applications and specialized fields. Many recent in vitro and in vivo studies have attempted to explain the biological mechanisms involved in PG-induced tissue regeneration/reparation. However, it remains unclear which parameters should be used in clinical applications to obtain satisfactory results in the healing of wounds. Toward this end, the present study focused on understanding the relationship between platelet concentrations and the cellular parameters of the cell types, i.e., fibroblasts, involved in wound healing. Normal human dermal fibroblasts were treated with PG-released supernatant at various concentrations in different assays (proliferation, migration, invasion, and in vitro scratch wound closure) to identify the most effective concentration to promote the fibroblasts' activities. Different concentrations of platelets per microliter in PG have different levels of efficacy in inducing fibroblast activity. The most effective concentration was obtained from PG at a concentration of approximately 0.5-1.5 × 10(6)  plt/μL; higher concentrations were less effective. This study shows that excessively high concentrations of platelets per microliter have an inhibitory effect on the wound healing processes and are, therefore, counterproductive.

  9. Long-term effect of platelet lysate on primary fibroblasts highlighted with a proteomic approach.

    PubMed

    Cipriani, Valentina; Ranzato, Elia; Balbo, Valeria; Mazzucco, Laura; Cavaletto, Maria; Patrone, Mauro

    2009-10-01

    The use of platelets and platelet derivatives has acquired clinical relevance as a means of accelerating wound healing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances able to regulate cellular activities. The purpose of this study was to evaluate the biological effects of platelet lysate (PL) on human primary skin fibroblasts. We studied cell viability, MAPK signalling and proteomic profile of fibroblasts exposed to a platelet lysate (PL) obtained from blood sample. Crystal violet and neutral red uptake assays showed the dose-response effects of PL on cell viability and metabolism at 3 and 6 days of exposure. Western blot demonstrated a more sustained activation of p38 than of ERK1/2. A proteomic approach was applied to identify soluble cellular components in primary fibroblasts that are differentially expressed in response to PL exposure. Protein identification was performed by mass spectrometry. The data demonstrate that human fibroblasts respond to PL exposure by modifying a number of proteins, related principally to stress response, metabolism and the cytoskeleton.

  10. The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts.

    PubMed

    Varkey, Mathew; Ding, Jie; Tredget, Edward E

    2014-12-01

    Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing.

  11. Effect of a volatile smoke component (acrolein) on human gingival fibroblasts: An in vitro study

    PubMed Central

    Anand, Nithya; Emmadi, Pamela; Ambalavanan, N.; Ramakrishnan, T.

    2011-01-01

    Aim: Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including human gingival fibroblasts. The aim of this present study was to estimate the effect of acrolein, a volatile fraction of cigarette smoke on the attachment, proliferation and ultra structure of human gingival fibroblasts in culture. Materials and Methods: Human gingival fibroblasts strains obtained from healthy subjects aged 20-30 years, were grown to confluency and utilized between 3rd -6th passages. The cell cultures seeded in 96 well microtitration plates at a density of 45,000 cells/well were incubated with acrolein at concentrations of 10-4, 3×10-5 and 10-5 . Attachment ability was evaluated after three hours using Neubauer hemocytometer. For the proliferation assay cell cultures seeded at a density of 10,000 cells/well were incubated at concentrations of 10-4, 3×10-5, 10-5, 3×10-6, 10-6 and cell count determined after 5 days using a hemocytometer. Cell morphology was examined under phase contrast microscope. Results: Acrolein produced a dose-dependent cytotoxic effect on human gingival fibroblasts with complete inhibition of attachment and proliferation at higher concentrations. Conclusion: This supports the hypothesis that cigarette smoke is a great risk factor in the development and progression of periodontal disease. PMID:22368362

  12. Effect of corrosion products (neodymium iron boron) on oral fibroblast proliferation.

    PubMed

    Evans, R D; McDonald, F

    1995-01-01

    The biological effects of the corrosion products of neodymium iron boron (Nd2Fe14B) magnets are largely unknown. The aim of this study was to identify the types of corrosion product and to evaluate the effect of the corrosion products (CP) of Nd2Fe14B magnets on the proliferation of human oral mucosal fibroblasts. Uncoated Nd2Fe14B magnets were stored in saline at 37 degrees C for 6 months and the corrosion products collected. 100 microL of a cell suspension (human oral mucosal fibroblasts [14 x 10(4) cells/mL]) was aliquoted into 72 wells of a 96-well plate, the remaining plates receiving culture medium only. After 12 h incubation at 37 degrees C, each well then received 100 microL of either (A) culture medium, (B) 100% CP, (C) 50% CP, or (D) 0% CP. The plates were reincubated at 37 degrees C for a further 48, 96, or 144 h. Fibroblast proliferation was assessed using the methylene blue uptake/elution technique. The compounds in the corrosion product were examined using quantitative X-ray analysis. Statistical analysis (ANOVA, Bonferroni's test 0.05, SAS v 6.04), showed that at each time point, the cell numbers in groups B, C, and D were significantly lower than group A. Within groups B, C, and D no significant differences were found, despite the suggestion of a dose response effect. Fibroblast proliferation in the presence of corrosion products was significantly lower than with culture medium. Fibroblast proliferation did occur in the presence of 0, 50, and 100% CP. The actual corrosion products appeared to be salts of iron but 3.2% (+/- 0.6) of neodymium chloride (NdCl3) was found.

  13. Effects of basic fibroblast growth factor on density and morphology of fibroblasts grown on root surfaces with or without conditioning with tetracycline or EDTA.

    PubMed

    Silvério, Karina G; Martinez, Aurora E T; Rossa, Carlos

    2007-09-01

    A study was conducted to evaluate in vitro the effect of root surface conditioning with basic fibroblast growth factor (b-FGF) on morphology and proliferation of fibroblasts. Three experimental groups were used: non-treated, and treated with 50 microg or 125 microg b-FGF/ml. The dentin samples in each group were divided into subgroups according to the chemical treatment received before application of b-FGF: none, or conditioned with tetracycline-HCl or EDTA. After contact with b-FGF for 5 min, the samples were incubated for 24 h with 1 ml of culture medium containing 1 x 10(5) cells/ml plus 1 ml of culture medium alone. The samples were then subjected to routine preparation for SEM, and random fields were photographed. Three calibrated and blind examiners performed the assessment of morphology and density according to two index systems. Classification and regression trees indicated that the root surfaces treated with 125 microg b-FGF and previously conditioned with tetracycline-HCl or EDTA presented a morphology more suggestive of cellular adhesion and viability (P = 0.004). The density of fibroblasts on samples previously conditioned with EDTA, regardless of treatment with b-FGF, was significantly higher than in the other groups (P < 0.001). The present findings suggest that topical application of b-FGF has a positive influence on both the density and morphology of fibroblasts.

  14. Biological effects of glycolic acid on dermal matrix metabolism mediated by dermal fibroblasts and epidermal keratinocytes.

    PubMed

    Okano, Yuri; Abe, Yumiko; Masaki, Hitoshi; Santhanam, Uma; Ichihashi, Masamitsu; Funasaka, Yoko

    2003-01-01

    Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

  15. 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.

  16. Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts

    PubMed Central

    Horiba, Minori; Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

    2017-01-01

    Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues and has emerged as a novel technology for medical applications. NTAPP affects cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the preconditioning effects of “mild PAM” which was prepared under relatively mild conditions, on fibroblasts against cellular injury generated by a high dose of hydrogen peroxide (H2O2). We observed the preconditioning effects of mild PAM containing approximately 50 μM H2O2. Hydrogen peroxide needs to be the main active species in mild PAM for it to exert preconditioning effects because the addition of catalase to mild PAM eliminated these effects. The nuclear translocation and recruitment of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response elements (ARE) in heme oxygenase 1 (HO-1) promoters and the up-regulation of HO-1 were detected in fibroblasts treated with mild PAM. The addition of ZnPP, a HO-1-specific inhibitor, or the knockdown of Nrf2 completely abrogated the preconditioning effects. Our results demonstrate that mild PAM protects fibroblasts from oxidative stress by up-regulating HO-1, and the H2O2-induced activation of the Nrf2-ARE pathway needs to be involved in this reaction. PMID:28169359

  17. Th17 cells favor inflammatory responses while inhibiting type I collagen deposition by dermal fibroblasts: differential effects in healthy and systemic sclerosis fibroblasts

    PubMed Central

    2013-01-01

    Introduction T helper (Th)-17 cells are increased in systemic sclerosis (SSc). We therefore assessed whether Th17 cells could modulate the inflammatory and fibrotic responses in dermal fibroblasts from healthy donors (HD) and SSc individuals. Methods Fibroblasts were obtained from 14 SSc and 8 HD skin biopsies. Th17 clones were generated from healthy peripheral blood upon enrichment of CC chemokine receptor (CCR)-4/CCR6/CD161 expressing cells. Their cytokine production was assessed by flow cytometry and multiplex beads immunoassay. Fibroblast production of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8, matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinase (TIMP)-1, MMP-2 and type-I collagen was quantified by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), and changes in their transcription levels assessed by real-time PCR. Intracellular signals were dissected by western blot and the use of pharmacological inhibitors. IL-17A, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) blocking reagents were used to assess the specificity of the observed effects. Results IL-17A increased MCP-1, IL-8 and MMP-1 production in a dose-dependent manner while having no effect on type I collagen in HD and SSc fibroblasts both at protein and mRNA levels. Nuclear factor-kappa B (NF-κB) and p38 were preferentially involved in the induction of MCP-1 and IL-8, while MMP-1 was most dependent on c-Jun N-terminal kinase (JNK). Supernatants of activated Th17 clones largely enhanced MCP-1, IL-8 and MMP-1 while strongly inhibiting collagen production. Of note, the production of MCP-1 and IL-8 was higher, while collagen inhibition was lower in SSc compared to HD fibroblasts. The Th17 clone supernatant effects were mostly dependent on additive/synergistic activities between IL-17A, TNF and in part IFN-γ. Importantly, the inhibition of type I collagen production induced by the Th17 clone supernatants was completely abrogated by

  18. Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Giampieri, Francesca; Afrin, Sadia; Mezzetti, Bruno; Quiles, Josè L; Bompadre, Stefano; Battino, Maurizio

    2017-01-21

    A protracted pro-inflammatory state is a major contributing factor in the development, progression and complication of the most common chronic pathologies. Fruit and vegetables represent the main sources of dietary antioxidants and their consumption can be considered an efficient tool to counteract inflammatory states. In this context an evaluation of the protective effects of strawberry extracts on inflammatory stress induced by E. coli LPS on human dermal fibroblast cells was performed in terms of viability assays, ROS and nitrite production and biomarkers of oxidative damage of the main biological macromolecules. The results demonstrated that strawberry extracts exerted an anti-inflammatory effect on LPS-treated cells, through an increase in cell viability, and the reduction of ROS and nitrite levels, and lipid, protein and DNA damage. This work showed for the first time the potential health benefits of strawberry extract against inflammatory and oxidative stress in LPS-treated human dermal fibroblast cells.

  19. Ca(2+)-dependent permeabilization of mitochondria and liposomes by palmitic and oleic acids: a comparative study.

    PubMed

    Belosludtsev, Konstantin N; Belosludtseva, Natalia V; Agafonov, Alexey V; Astashev, Maxim E; Kazakov, Alexey S; Saris, Nils-Erik L; Mironova, Galina D

    2014-10-01

    In the present work, we examine and compare the effects of saturated (palmitic) and unsaturated (oleic) fatty acids in relation to their ability to cause the Ca(2+)-dependent membrane permeabilization. The results obtained can be summarized as follows. (1) Oleic acid (OA) permeabilizes liposomal membranes at much higher concentrations of Ca(2+) than palmitic acid (PA): 1mM versus 100μM respectively. (2) The OA/Ca(2+)-induced permeabilization of liposomes is not accompanied by changes in the phase state of lipid bilayer, in contrast to what is observed with PA and Ca(2+). (3) The addition of Ca(2+) to the PA-containing vesicles does not change their size; in the case of OA, it leads to the appearance of larger and smaller vesicles, with larger vesicles dominating. This can be interpreted as a result of fusion and fission of liposomes. (4) Like PA, OA is able to induce a Ca(2+)-dependent high-amplitude swelling of mitochondria, yet it requires higher concentrations of Ca(2+) (30 and 100μM for PA and OA respectively). (5) In contrast to PA, OA is unable to cause the Ca(2+)-dependent high-amplitude swelling of mitoplasts, suggesting that the cause of OA/Ca(2+)-induced permeability transition in mitochondria may be the fusion of the inner and outer mitochondrial membranes. (6) The presence of OA enhances PA/Ca(2+)-induced permeabilization of liposomes and mitochondria. The paper discusses possible mechanisms of PA/Ca(2+)- and OA/Ca(2+)-induced membrane permeabilization, the probability of these mechanisms to be realized in the cell, and their possible physiological role.

  20. Immunomodulatory Effects of Bee Venom in Human Synovial Fibroblast Cell Line

    PubMed Central

    Mohammadi, Ebrahim; Vatanpour, Hossein; H Shirazi, Farshad

    2015-01-01

    As in Iranian traditional medicine, bee venom (BV) is a promising treatment for the rheumatoid arthritis (RA) which is considered as a problematic human chronic inflammatory disease in the present time. Smoking is considered to be a major risk factor in RA onset and severity. The main aim of this study is to investigate the effects of BV on cigarette smoke-induced inflammatory response in fibroblast-like synoviocytes (FLS). Cytotoxicity of cigarette smoke condensate (CSC) and bee venom were determined by the tetrazolium (MTT) method in cultured synovial fibroblastes. The expression of interleukin-1β and sirtuin1 mRNA were analyzed by SYBR green real-time quantitative PCR. Differences between the mean values of treated and untreated groups were assessed by student t-test. Based on MTT assay, CSC and BV did not exert any significant cytotoxic effects up to 40 µg/mL and 10 µg/mL, respectively. Our results showed that interleukin-1β mRNA level was significantly up-regulated by CSC treatments in LPS-stimulated synoviocytes in a dose-dependent manner. Conversely, the expressions of IL-1β and Sirt1 were up-regulated even in lower concentrations of BV and attenuated at higher concentrations. Also, BV attenuated the CSC-induced and LPS-induced inflammatory responses in synovial fibroblasts. Our results support the epidemiological studies indicating pro-inflammatory effects of CSC and anti-inflammatory effects of BV on FLS cell line. PMID:25561937

  1. Lithium effects on circadian rhythms in fibroblasts and suprachiasmatic nucleus slices from Cry knockout mice.

    PubMed

    Noguchi, Takako; Lo, Kevin; Diemer, Tanja; Welsh, David K

    2016-04-21

    Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3β (GSK-3β), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3β. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

  2. [Radioprotective effect of helium-neon laser radiation for fibroblast cells].

    PubMed

    Voskanian, K Sh; Mitsyn, G V; Gaevskiĭ, V N

    2007-01-01

    Effects of combined exposure to 633-nm laser waves and gamma-radiation, and laser waves and protons with the energy of 150 MeV on survivablilty of mice fibroblast cells C3H10T1/2 were compared. Cell suspension (1 - 5 x 10(5) cells/ml) was distributed in 2-ml plastic vials with 1 cm in diameter time interval between two exposures in a combination was no more than 60 s. immediately after exposure a required quantity of cells was inoculated in special vials for survivability assessment. Based on results of the experiment, preliminary and repeated laser treatment was favorable to survivability of fibroblast cells subjected to gamma- or proton irradiation (dose variation factor was within 1.3 to 2.2). Simultaneous exposure of C3H10T1/2 cells to the laser and proton beams also increased their survivability. The radioprotective effect of the helium-neon laser on fibroblasts earlier exposed to ionizing radiation is of chief interest, as most of the present-day radioprotectors are effective only if introduced into organism prior to exposure.

  3. 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.

  4. 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.

  5. Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

    PubMed

    Chang, Y C; Hu, C C; Tseng, T H; Tai, K W; Lii, C K; Chou, M Y

    2001-09-01

    Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

  6. Biological effects of cobalt-chromium nanoparticles and ions on dural fibroblasts and dural epithelial cells.

    PubMed

    Behl, Bharat; Papageorgiou, Iraklis; Brown, Christopher; Hall, Richard; Tipper, Joanne L; Fisher, John; Ingham, Eileen

    2013-05-01

    The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses.

  7. Effect of Basic Fibroblast Growth Factor on Achilles Tendon Healing in Rabbit

    PubMed Central

    Najafbeygi, Arash; Fatemi, Mohammad Javad; Lebaschi, Amir Hussein; Mousavi, Seyed Jaber; Husseini, Seyed Abouzar; Niazi, Mitra

    2017-01-01

    BACKGROUND Tendon injuries are common and it takes a long time for an injured tendon to heal. Adverse phenomena such as adhesion and rupture are associated with these injuries. Finding a method to reduce the time required for healing which improves the final outcome, will lead to decreased frequency and intensity of adverse consequences. This study was designed to investigate the effects of basic fibroblast growth factor on the healing of the Achilles tendon in rabbits METHODS In 10 New Zealand white rabbits, Achilles tendon was cut at the intersection of the distal and middle thirds on both hind legs. One microgram of recombinant basic fibroblast growth factor (bFGF) was injected in the proximal and distal stumps of the cut tendon on the right side (study group). Normal saline of equal volume was injected on the left side in the same way (control group). Then the tendons were repaired with 5/0 nylon using modified Kessler technique. A cast was made to immobilize each leg. On day 42, rabbits were euthanized and both hind legs were amputated. Tensometry and histopathologic examination were done on specimens. RESULTS In tensometric studies, more force was required to rupture the repair site in study group. In histopathologic examination, collagen fibers had significantly better orientation and organization in the study group. No difference was noted regarding number of fibroblast and fibrocytes, and degree of angiogenesis in the two groups. CONCLUSION Application of basic fibroblast growth factor at tendon repair site improves the healing process through improvement of collagen fiber orientation and increase in biomechanical resistance. PMID:28289610

  8. Effect of time of extraction on the biocompatibility of endodontic sealers with primary human fibroblasts.

    PubMed

    Scelza, Miriam Zaccaro; Coil, Jeffrey; Alves, Gutemberg Gomes

    2012-01-01

    The aim of this work was to evaluate the effects of different times of extraction on the cytotoxicity of six representatives of different root canal sealer groups-Real Seal SE, AH Plus, GuttaFlow, Sealapex, Roth 801, and ThermaSeal Plus-with human gingival fibroblasts. The materials were prepared according to manufacturers' specifications, and were incubated in culture medium (DMEM) at 37ºC for 1, 7, 14, 21, and 28 days, with daily washing, to simulate periodontal ligament clearance. Human fibroblasts were exposed to the final extracts at 24 hours, and cell viability was determined by MTT assay, with exposure to unconditioned DMEM as a negative control. Statistical analysis comparing cytotoxicities at each exposure time was performed by ANOVA with Scheffé adjustment for multiple comparisons at a 95% confidence level. Results indicated that GuttaFlow was significantly less cytotoxic than all other sealers (p < 0.05) at 1 day of extraction. After 7 days of extraction, cell viability for GuttaFlow was significantly increased as compared with that of all groups except sealer AH Plus. At day 14, cytotoxicity of Sealapex was significantly higher than that of all other sealers (p < 0.05). At days 21 and 28, there were no significant differences in cytotoxicity among sealer groups. All materials presented some level of cytotoxicity to fibroblasts, while GuttaFlow was the least cytotoxic sealer tested. However, the cytotoxicity of all materials seemed to decrease similarly in a time-dependent manner.

  9. Sequence analysis of bovine C/EBPδ gene and its adipogenic effects on fibroblasts.

    PubMed

    Wang, Hong; Cheng, Gong; Fu, Changzhen; Wang, Hongbao; Yang, Wucai; Wang, Hongcheng; Zan, Linsen

    2014-01-01

    CCAAT/enhancer binding protein delta (C/EBPδ), an important transcriptional factor, regulates cell growth, differentiation and adipogenesis in humans and mice. However, we lack of directive information on the effects of C/EBPδ gene in bovine cells. In the present study, we cloned the CDS areas of bovine C/EBPδ gene and predicted its sequence characteristics. Moreover, we constructed the recombinant adenovirus plasmids of bovine C/EBPδ gene and harvested the subsequent adenoviruses to infect bovine primary fibroblasts. Oil Red O staining results showed lipid droplets accumulated gradually in the adenoviruses treated fibroblasts. Time course real-time PCR results indicated that over-expression of exogenous C/EBPδ regulated the mRNA expression levels of some key adipogenic genes, herein, activated the C/EBPα expression, increased lipoprotein lipase and fatty acid binding protein 4 mRNA expression levels, whereas inhibited leptin receptor gene. In conclusion, the present study demonstrates that the elevated C/EBPδ can induce the adipogenesis in the fibroblasts of cattle.

  10. Effects of Hypoxia, Surrounding Fibroblasts, and p16 Expression on Breast Cancer Cell Migration and Invasion.

    PubMed

    Zhang, Jun; Li, Liyuan; Lu, Yi

    2015-01-01

    Cancer cell migration and invasion play essential roles in the metastatic cascade that transforms the local, noninvasive confined tumor cells to the motile, metastatic cancer cells moving through the extracellular matrix and basement into the circulation. Accumulated evidences suggest that intratumoral hypoxia, a characteristic of fast-growing solid tumors, promotes cancer cell motile and invasive abilities. In this study, we investigated the effects of hypoxia, surrounding fibroblasts, and p16 expression on the migration and invasion of breast cancer cells. We found that hypoxia promoted breast cancer cell migration and invasion, and cocultured fibroblasts stimulated invasiveness of breast cancer cells. Moreover, by using a Tet-on inducible system, we found that p16 is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells. These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia-mediated cancer cell migration and invasion. This study may provide important validation for p16-mediated cancer therapy either by gene therapy or pharmacological activation of internal p16 gene that is usually inactive due to hypermethylation in the tumor cells.

  11. 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

  12. 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

  13. Adverse effects of citrate/gold nanoparticles on human dermal fibroblasts.

    PubMed

    Pernodet, Nadine; Fang, Xiaohua; Sun, Yuan; Bakhtina, Asya; Ramakrishnan, Aditi; Sokolov, Jonathan; Ulman, Abraham; Rafailovich, Miriam

    2006-06-01

    Nanoscale engineering is one of the most dynamically growing areas at the interface between electronics, physics, biology, and medicine. As there are no safety regulations yet, concerns about future health problems are rising. We investigated the effects of citrate/gold nanoparticles at different concentrations and exposure times on human dermal fibroblasts. We found that, as a result of intracellular nanoparticle presence, actin stress fibers disappeared, thereby inducing major adverse effects on cell viability. Thus, properties such as cell spreading and adhesion, cell growth, and protein synthesis to form the extracellular matrix were altered dramatically. These results suggest that the internal cell activities have been damaged.

  14. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

    PubMed

    Alakomi, H L; Skyttä, E; Saarela, M; Mattila-Sandholm, T; Latva-Kala, K; Helander, I M

    2000-05-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

  15. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

    PubMed Central

    Alakomi, H.-L.; Skyttä, E.; Saarela, M.; Mattila-Sandholm, T.; Latva-Kala, K.; Helander, I. M.

    2000-01-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl2. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances. PMID:10788373

  16. Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

    PubMed

    Ozawa, Yumi; Hayashi, Shujiro; Hamasaki, Yoichiro; Hatamochi, Atsushi

    2016-12-01

    Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5 × 10(-7) to 5 × 10(-3) M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of (3)H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of (3)H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5 × 10(-7) to 5 × 10(-4)M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5 × 10(-7) and 5 × 10(-3) M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect

  17. Toxic effects of various retrograde root filling materials on gingival fibroblasts and rat sarcoma cells.

    PubMed

    Peltola, M; Salo, T; Oikarinen, K

    1992-06-01

    The aim of this in vitro study was to evaluate the effect of amalgam, glass ionomer, composite and titanium on the growth of gingival fibroblasts (GF) and rat sarcoma cells (UMR) in vitro. The cells were either obtained from gingival biopsies taken during deliberation of unerupted canines (GF) or were of commercial origin (UMR). Equal numbers of cells were placed on culture dishes and incubated for a period of two weeks with the freshly prepared test materials. The cultures were photographed through a light microscope after 7 days incubation and finally counted after 14 days. It was shown that the proliferation of gingival fibroblasts was less disturbed by titanium, being approximately 96% of the control value (cell cultures without test particles), followed by composite, amalgam and glass ionomer (61%, 49% and 35% of the control value respectively). The number of UMR cells after 14 days incubation with the various materials was 76% of the control value with titanium, 12% with composite and 5% with both amalgam and glass ionomer. Inhibition of cell growth (UMR) around the test particles was most prominent around amalgam and glass ionomer, followed by composite and titanium. These effects were noted only with freshly prepared components however, so that the toxic reaction was less pronounced or minimal in a second incubation using the same particles sterilized in between. The results demonstrated that potential retrograde root filling materials have a variable toxic effect on gingival fibroblasts and rat sarcoma cells. The fact that the influence on proliferation disappeared when the test was performed with materials already tested once may be of clinical importance when estimating the biocompatibility in vivo.

  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. 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

  20. 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

  1. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization.

    PubMed

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

    2016-01-26

    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.

  2. Effect of low-intensity pulsed ultrasound on l929 fibroblasts

    PubMed Central

    Franco de Oliveira, Rodrigo; Pires Oliveira, Deise A. A.; Soares, Cristina Pacheco

    2011-01-01

    Introduction Ultrasound has proven to be an important therapeutic resource regarding musculoskeletal disease and is routinely used in physical therapy and medicine both therapeutically and diagnostically. The aim of the present study was to analyse the effects with different ultrasound intensities in order to establish the ideal radiation level in cell cultures. Material and methods Fibroblast cell cultures were divided into five groups: group I – control (did not receive irradiation); group II – 0.2 W/cm2 in pulsed mode at 10% (1 : 9 duty cycle); group III – 0.6 W/cm2 in pulsed mode at 10% (1 : 9 duty cycle); group IV – 0.2 W/cm2 in pulsed mode at 20% (2 : 8 duty cycle); and group V – 0.6 W/cm2 in pulsed mode at 20% (2 : 8 duty cycle). Each group was irradiated with 24-h intervals, observing the following post-irradiation incubation times: 24, 48, 72 and 96 h; after 24 h of each irradiation, cultures were analysed using the MTT method. Results Analysis of the results following ultrasound irradiation demonstrated that the effect of ultrasound with 0.6 W/cm2 in pulsed mode at 10% (1 : 9 duty cycle) was statistically significant in relation to ultrasonic irradiation in pulsed mode at 20% (2 : 8 duty cycle) (p < 0.05). Conclusions According to parameters used in the irradiation of cultivated fibroblasts, the pulse mode regime and the control of intensity are of fundamental importance for the optimal use of therapeutic ultrasound. Furthermore, low and medium intensities decreased cell damage, which establishes that acoustic pulsed energy induces the proliferation of fibroblast cells. PMID:22291760

  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. 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.

  5. A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion.

    PubMed

    Coppe, Jean-Philippe; Boysen, Megan; Sun, Chung Ho; Wong, Brian J F; Kang, Mo K; Park, No-Hee; Desprez, Pierre-Yves; Campisi, Judith; Krtolica, Ana

    2008-07-01

    Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.

  6. Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists

    PubMed Central

    Chaïb, N; Kabré, E; Alzola, E; Pochet, S; Dehaye, J P

    2000-01-01

    The permeabilizing effect of P2X7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37°C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 μM) and it was decreased when the temperature was lowered to 25°C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 μM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A2 (iPLA2). Bromoenol lactone (BEL) inhibited the iPLA2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X7 receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor. PMID:10683195

  7. 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

  8. Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells

    PubMed Central

    Kim, Sangwoo; Jeon, Sangmi; Hui, Zheng; Kim, Young; Im, Yeonggwan; Lim, Wonbong; Kim, Changsu; Choi, Hongran; Kim, Okjoon

    2015-01-01

    Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells. PMID:25662537

  9. Ameliorative effects of Eriobotrya japonica seed extract on cellular aging in cultured rat fibroblasts.

    PubMed

    Muramoto, Kazuyo; Quan, Rong-Dan; Namba, Toshiharu; Kyotani, Shojiro; Miyamura, Mitsuhiko; Nishioka, Yutaka; Tonosaki, Keiichi; Doi, Yoshinori L; Kaba, Hideto

    2011-04-01

    To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

  10. Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

    PubMed Central

    Santangelo, Kelly S; Johnson, Amanda L; Ruppert, Amy S; Bertone, Alicia L

    2007-01-01

    Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and

  11. 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.

  12. Effects of mebendazole on protein biosynthesis and secretion in human-derived fibroblast cultures.

    PubMed

    Soto, H; Massó, F; Cano, S; Díaz de León, L

    1996-07-26

    Previous results of our group revealed that mebendazole, a broad spectrum anthelmintic drug with antimicrotubular properties, used for the treatment of liver cirrhosis, decreased total collagen content and biosynthesis in liver upon treatment. In the present study, we have evaluated the effects of mebendazole (5-50 micrograms/mL) on protein synthesis, secretion, and deposition in human-derived fibroblast cultures. The results showed a decrease in cell viability (18.5 +/- 0.9%) at 50 micrograms/mL. [3H]Thymidine incorporation diminished gradually with increasing mebendazole concentrations, reaching a plateau (53.67%) between 30 and 50 micrograms/mL. In late logarithmic phase cultures, the drug caused a decrease of [3H]proline incorporation (43.10%) and collagen biosynthesis (58.61%) in the extracellular matrix. This correlated with an increase in radioactivity in total proteins (51.28%) of the intracellular fraction. Similar results were obtained when mebendazole was assayed in post-confluent fibroblast cultures. The electrophoretic patterns of the extracellular matrix showed a decrease of radioactive collagenous components (alpha chains and beta dimers). By contrast, in the intracellular fraction an increase of radioactive collagen precursors (pro alpha chains) was observed. Immunofluorescence studies and immunotransfer analysis, using polyclonal anti-type I collagen antibodies, revealed an accumulation of intracellular collagen which included: collagen pro alpha chains, alpha chains, and low molecular weight peptides. The results obtained suggest that mebendazole interferes with the transcellular mobilization of proteins, resulting in a decrease of secretion and deposition of extracellular matrix proteins, and an accumulation of intracellular collagenous components. The intracellular accumulation of newly synthesized proteins could cause a feedback regulation in fibroblast cultures.

  13. 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.

  14. Membrane Deformation and Permeabilization Caused by Microplasma Irradiation

    NASA Astrophysics Data System (ADS)

    Motomura, Hideki; Nagaiwa, Hidenori; Yamamoto, Kenta; Kido, Yugo; Ikeda, Yoshihisa; Satoh, Susumu; Jinno, Masafumi

    2016-09-01

    The microplasma irradiation achieves high gene taransfection efficiency and high cell survivability simultaneously. For this purpose, we have developed a special plasma source using a microcapillary electrode. However, it is not clear how the stimuli of effective factors generated by plasma, such as current, charge, field, chemical species, cause transfection. In this study, we used artificial cell which is a spherical vesicle consisting of a lipid bilayer to visualize membrane dynamics and permeabilization caused by microplasma irradiation. Dioleoyl phosphatidylcholine (DOPC) was used as phospholipid molecules forming the lipid bilayer. The artificial cells were prepared by natural swelling method. Fluorescent labeled polyethylene glycol (PEG) polymers (Nanocs, MPEG Fluorescein, MW = 1000) were encapsulated in the artificial cells. The artificial cells were exposed to the microplasma for 5 ms and 10-20% of decrease of the dye fluorescence in the artificial cells was observed. This result suggests the outflow of the MPEG polymers through temporary poration or deformation of the lipid bilayer. The membrane deformation dynamics was directly observed with a microscope and the relationship to the polymer outflow will be shown at the conference. This work was partly supported by a Grant-in-Aid (25108509 and 15H00896) from JSPS and a grant from Ehime University.

  15. Vector-free intracellular delivery by reversible permeabilization

    PubMed Central

    Annibaldi, Valeria; Gallagher, Louise; Mulholland, Joanne; Molloy, Emer L.; Breen, Conor J.; Gilbert, Jennifer L.; Martin, Darren S.; Maguire, Michael; Curry, Fitz-Roy

    2017-01-01

    Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications. PMID:28358921

  16. Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

    PubMed Central

    Fernandez, M P; Correa, J U; Cabib, E

    1982-01-01

    Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment. Images PMID:6216245

  17. Additive and synergistic membrane permeabilization by antimicrobial (lipo)peptides and detergents.

    PubMed

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

    2014-05-20

    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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

    NASA Astrophysics Data System (ADS)

    Shih, Chi-Jen; Lu, Pei-Shan; Hsieh, Chih-Hsin; Chen, Wen-Cheng; Chen, Jian-Chih

    2014-09-01

    The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity.

  5. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

    SciTech Connect

    Layman, D.L.; Diedrich, D.L.

    1987-06-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by /sup 3/H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in /sup 3/H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin.

  6. Effect of Cyclosporin A and Angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts

    PubMed Central

    Supraja, Ajitkumar; Dinesh, Murugan Girija; Rajasekaran, Subbarayan; Balaji, Thodur Madapusi; Rao, Suresh Ranga

    2016-01-01

    Background: To evaluate the effect of Cyclosporin A (CsA) and angiotensin II (Ang II) on cytosolic calcium levels in cultured human gingival fibroblasts (HGFs). Materials and Methods: Healthy gingival samples from six volunteers were obtained, and primary HGFs were cultured. Cell viability and proliferation assay were performed to identify the ideal concentrations of CsA and Ang II. Cytosolic calcium levels in cultured gingival fibroblasts treated with CsA and Ang II were studied using colorimetric assay, confocal and fluorescence imaging. Statistical analyses were done using SPSS software and GraphPad Prism. Results: Higher levels of cytosolic levels were evident in cells treated with CsA and Ang II when compared to control group and was statistically significant (P < 0.05) in both colorimetric assay and confocal imaging. Fluorescent images of the cultured HGFs revealed the same. Conclusion: Thus calcium being a key player in major cellular functions, plays a major role in the pathogenesis of drug-induced gingival overgrowth. PMID:27857765

  7. Treatment effect of coenzyme Q(10) and an antioxidant cocktail in fibroblasts of patients with Sanfilippo disease.

    PubMed

    Matalonga, Leslie; Arias, Angela; Coll, María Josep; Garcia-Villoria, Judit; Gort, Laura; Ribes, Antonia

    2014-05-01

    Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 μmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.

  8. Small molecular antioxidants effectively protect from PUVA-induced oxidative stress responses underlying fibroblast senescence and photoaging.

    PubMed

    Briganti, Stefania; Wlaschek, Meinhard; Hinrichs, Christina; Bellei, Barbara; Flori, Enrica; Treiber, Nicolai; Iben, Sebastian; Picardo, Mauro; Scharffetter-Kochanek, Karin

    2008-09-01

    Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.

  9. Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts.

    PubMed

    Khammanit, R; Chantakru, S; Kitiyanant, Y; Saikhun, J

    2008-07-01

    The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.

  10. Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay.

    PubMed

    Rose, Michael T

    2012-08-01

    The objective of this study was to determine the effect of platelet derived growth factor BB (PDGF), epidermal growth factor (EGF), transforming growth factor β1 (TGFβ1), insulin like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) on the proliferation and migration of equine oral mucosa and leg skin fibroblast cell lines, using an in vitro scratch assay. Fibroblasts from the two sites were firstly grown to confluence and then an area of cells removed (cell void area). Cell migration alone (with the addition of the mitosis inhibitor mitomycin-C to the culture media) and proliferation and migration combined (without mitomycin-C) into the cell void area were observed at 0, 5, 10, 24 and 36 h. The presence of mitomycin-C in the culture media significantly slowed the closure of the cell void area, as mitosis was inhibited. For the oral cells only, TGFβ1 significantly slowed both migration (with mitomycin-C) and proliferation and migration combined (without mitomycin-C). For the limb cells only, both PDGF and FGF-2 significantly increased fibroblast proliferation and migration combined (without mitomycin-C). For both cell types, EGF significantly reduced migration (with mitomycin-C). IGF-1 had no effect on any of the parameters measured. It was concluded that TGFβ1, PDGF and FGF-2 have differential effects on the proliferation and migration of equine oral and limb fibroblasts. These differences in fibroblast responses to growth factors may in part form the basis of the different clinical outcomes for oral and limb wounds.

  11. Additive anti-inflammatory effect of formoterol and budesonide on human lung fibroblasts

    PubMed Central

    Spoelstra, F; Postma, D; Hovenga, H; Noordhoek, J; Kauffman, H

    2002-01-01

    Background: It has been shown that treatment with a long acting ß2 agonist in addition to a glucocorticoid is beneficial in the treatment of asthma. In asthma inflammatory cells, particularly eosinophils, migrate into the pulmonary tissue and airway lumen by means of adhesion molecules expressed on resident tissue cells—that is, fibroblasts—and become activated by cytokines and adhesive interactions. A study was undertaken to determine whether an interaction exists between the long acting ß2 agonist formoterol and the glucocorticoid budesonide on inhibition of adhesion molecule expression, as well as chemo/cytokine production by human lung fibroblasts. Methods: Lung fibroblasts were preincubated with therapeutically relevant drug concentrations of 10-8 M to 10-10 M. Cells were stimulated with interleukin (IL)-1ß (1 or 10 U/ml) for 8 hours and supernatants were collected for measurement of GM-CSF and IL-8 concentrations. The cells were fixed and subjected to a cell surface ELISA technique to measure the expression of ICAM-1 and VCAM-1. Results: Formoterol exerted an additive effect on the inhibition of IL-1ß stimulated ICAM-1 and VCAM-1 upregulation and GM-CSF production by budesonide in concentrations of 10-9 M and above (p<0.05). IL-8 production was not influenced by formoterol. Conclusion: Formoterol exerts an additive effect on the anti-inflammatory properties of budesonide. In vitro data support the finding that the combination of budesonide and formoterol in asthma treatment strengthens the beneficial effect of either drug alone. PMID:11867828

  12. 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.

  13. CCN2 and CCN5 exerts opposing effect on fibroblast proliferation and transdifferentiation induced by TGF-β.

    PubMed

    Xu, Honghai; Li, Peng; Liu, Mengting; Liu, Cong; Sun, Zhengming; Guo, Xiong; Zhang, Yuelin

    2015-11-01

    Epidural fibrosis might occur after lumbar discectomy and contributes to failed back syndrome. Transforming growth factor (TGF)-β has been reported to influence multiple organ fibrosis, in which connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 2 (CCN2) and CCN5 are involved. However, the effect of CCN2 and CCN5 on TGF-β induced fibrosis has not yet been elucidated. This study reports that CCN2 and CCN5 play opposing roles in cell proliferation and transdifferentiation of human skin fibroblasts or rabbit epidural scar-derived fibroblasts exposed to TGF-β. We observed that TGF-β1 induced fibroblasts proliferation and differentiation in a dose-dependent manner (from 0 μg/L to 20 μg/L). Meanwhile, CCN2 expression is up-regulated while CCN5 expression is inhibited by TGF-β1 exposure. Furthermore, it is demonstrated that CCN2 overexpression leads to promoted proliferation and elevated collagen and α-smooth muscle actin (α-SMA) expression, which are inhibited by CCN5 overexpression. Moreover, it is shown that the cysteine knot (CT) domain, present in CCN2 but absent in CCN5, plays an essential part in fibroblast proliferation and differentiation. Additionally, enhanced TGF-β and CCN2 expression but decreased CCN5 expression is found in rabbit epidural scar-derived fibroblasts. Overall, the results show the opposing effects of CCN2 and CCN5 on fibroblast proliferation and transdifferentiation induced by TGF-β.

  14. Effects of hepatocyte growth factor on MMP-2 expression in scleral fibroblasts from a guinea pig myopia model

    PubMed Central

    Li, Xiu-Juan; Yang, Xiao-Peng; Wan, Guang-Ming; Wang, Yu-Ying; Zhang, Jin-Song

    2014-01-01

    AIM To investigate the effects of hepatocyte growth factor (HGF) on MMP-2 expression in scleral fibroblasts from guinea pig with LIM. METHODS Sixty 1-week-old guinea pigs were chosen for the study. The right eyes were treated with -10.0 D lenses as the LIM group; the left eyes remained untreated as the control group. The refraction and axial length were measured by streak retinoscopy and A-scan ultrasonography respectively prior to and 4 weeks after the experiment. Four weeks later, the guinea pigs were sacrificed and primary scleral fibroblasts were taken for tissue culture. The 3rd-5th generation scleral fibroblasts were chosen for the experiments. The expression levels of HGF and MMP-2 protein in the scleral fibroblasts were analyzed by Western blotting. After HGF with different doses acted on the scleral fibroblasts of the control group, MMP-2 protein expression in the scleral fibroblasts was analyzed by Western blotting. HGF siRNA was transfected into the scleral fibroblasts of the LIM group and the protein expressions of HGF and MMP-2 were analyzed by Western blotting. RESULTS The LIM group became myopic with a significant increase in axial length (7.97±0.29 mm vs 7.01±0.26 mm, P<0.05), and a significant decrease in refraction (-5.06±0.31 D vs 0.55±0.25 D, P<0.05) compared with the control group. The protein expression of HGF in the scleral fibroblasts of the LIM group was significantly higher compared with the control group ( 1.26±0.04 vs 0.32 ±0.04, P<0.05). The protein expression of MMP-2 in the scleral fibroblasts of the LIM group was significantly higher compared with the control group (0.89±0.06 vs 0.42±0.05, P<0.05). In the scleral fibroblasts of the control group, HGF(0, 0.1, 1, 10 ng/mL) upregulated MMP-2 protein expression in a dose-dependent manner (0.35±0.03, 0.44±0.02, 0.91±0.03, 1.33±0.04, all P<0.05). In the scleral fibroblasts of the LIM group transfected with HGF siRNA, MMP-2 protein expressions were significantly decreased

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

    PubMed

    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.

  16. The Effects of Ascorbate, N-Acetylcysteine, and Resveratrol on Fibroblasts from Patients with Mitochondrial Disorders

    PubMed Central

    Douiev, Liza; Soiferman, Devorah; Alban, Corinne; Saada, Ann

    2016-01-01

    Reactive oxygen species (ROS) are assumed to be implicated in the pathogenesis of inborn mitochondrial diseases affecting oxidative phosphorylation (OXPHOS). In the current study, we characterized the effects of three small molecules with antioxidant properties (N-acetylcysteine, ascorbate, and resveratrol) on ROS production and several OXPHOS parameters (growth in glucose free medium, ATP production, mitochondrial content and membrane potential (MMP)), in primary fibroblasts derived from seven patients with different molecularly defined and undefined mitochondrial diseases. N-acetylcysteine appeared to be the most beneficial compound, reducing ROS while increasing growth and ATP production in some patients’ cells. Ascorbate showed a variable positive or negative effect on ROS, ATP production, and mitochondrial content, while incubation with resveratrol disclosed either no effect or detrimental effect on ATP production and MMP in some cells. The individual responses highlight the importance of investigating multiple parameters in addition to ROS to obtain a more balanced view of the overall effect on OXPHOS when evaluating antioxidant treatment options for mitochondrial diseases. PMID:28025489

  17. Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species-induced cell death: a comparative study of H₂O₂, paraquat, t-BHP, etoposide and TNF-α-induced cell death.

    PubMed

    Rincheval, Vincent; Bergeaud, Marie; Mathieu, Lise; Leroy, Jacqueline; Guillaume, Arnaud; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc

    2012-08-01

    In this study, we have compared several features of cell death triggered by classical inducers of apoptotic pathways (etoposide and tumour necrosis factor (TNF)-α) versus exogenous reactive oxygen species (ROS; hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (t-BHP)) or a ROS generator (paraquat). Our aim was to characterize relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on source and identity of ROS. First, we have found that these five inducers trigger oxidative stress, mitochondrial membrane permeabilization (MMP), cytochrome c (cyt c) release from mitochondria and cell death. In each case, cell death could be inhibited by several antioxidants, showing that it is primarily ROS dependent. Second, we have highlighted that during etoposide or TNF-α treatments, intracellular ROS level, MMP and cell death are all regulated by caspases and Bcl-2, with caspases acting early in the process. Third, we have demonstrated that H₂O₂-induced cell death shares many of these characteristics with etoposide and TNF-α, whereas t-BHP induces both caspase-dependent and caspase-independent cell death. Surprisingly, paraquat-induced cell death, which harbours some characteristics of apoptosis such as cyt c release and caspase-3 activation, is not modulated by Bcl-2 and caspase inhibitors, suggesting that paraquat also triggers non-apoptotic cell death signals. On the one hand, these results show that endogenous or exogenous ROS can trigger multiple cell death pathways with Bcl-2 and caspases acting differentially. On the other hand, they suggest that H₂O₂ could be an important mediator of etoposide and TNF-α-dependent cell death since these inducers trigger similar phenotypes.

  18. 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.

  19. Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

    PubMed

    Pasi, Francesca; Sanna, Samuele; Paolini, Alessandro; Alquati, Marco; Lascialfari, Alessandro; Corti, Maurizio Enrico; Liberto, Riccardo Di; Cialdai, Francesca; Monici, Monica; Nano, Rosanna

    2016-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

  20. 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

  1. A permeabilized cell system that assembles filamentous bacteriophage

    PubMed Central

    Feng, Jian-nong; Russel, Marjorie; Model, Peter

    1997-01-01

    A permeabilized cell system has been developed that is capable of assembling filamentous phage only upon addition of exogenous thioredoxin. The in vitro system exhibits the same component requirements seen in vivo: functional thioredoxin, an intact packaging signal in the substrate DNA, and the assembly protein, pIV. This crude in vitro system is insensitive to inhibitors of protein or DNA synthesis, demonstrating that particle assembly uses components that had accumulated before cell permeabilization. The temporal separation of the synthetic period, during which phage proteins and DNA accumulate, from the assembly period enabled us to examine the energy requirement for assembly. We show here that ATP hydrolysis is required for filamentous phage assembly and that the proton motive force is also important. PMID:9108106

  2. Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells.

    PubMed

    Kehlenbach, Ralph H; Port, Sarah A

    2016-01-01

    Nuclear protein import and export assays in permeabilized cells have been instrumental for the identification of transport factors and for the molecular characterization of nucleocytoplasmic transport pathways. Our original assay to quantitatively analyze CRM1-dependent export was based on stably transfected cells expressing GFP-NFAT. We now present a simplified version of the assay using transiently transfected cells expressing GFP-NFAT or GFP-snurportin1 as a fluorescent export cargo and mCherry-emerin as a marker protein for transfected cells. CRM1- and Ran-dependent export is recapitulated in digitonin-permeabilized cells and quantified by flow cytometry. The assay should be applicable to other combinations of cargo and marker proteins.

  3. Hydrolysis of whey lactose using CTAB-permeabilized yeast cells.

    PubMed

    Kaur, Gurpreet; Panesar, Parmjit S; Bera, Manav B; Kumar, Harish

    2009-01-01

    Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.

  4. 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

  5. The effects of solcoseryl on the growth and multiplication of chick embryo fibroblasts cultivated "in vitro".

    PubMed

    Brasseur, R; De Paermentier, F

    1979-01-01

    The action of Solcoseryl, a free protein extract of calf blood, was studied on chick embryo fibroblasts cultivated in vitro. Solcoseryl stimulates the permitotic DNA synthesis and increases the number of mitoses.,

  6. The effect of flavonoids on transduction mechanisms in lipopolysaccharide-treated human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Jiménez-Estrada, Manuel; Maldonado, Silvia

    2007-09-01

    Periodontal disease comprises a group of infections that lead to inflammation of the gingival and destruction of periodontal tissues and is accompanied by the loss of the alveolar bone with eventual exfoliation of the teeth. Porphyromonas gingivalis is a Gram-negative bacteria obtained from the periodontal pocket of patients with aggressive and chronic periodontitis. This bacteria presents in the external membrane lipopolysaccharide (LPS). Flavonoids are molecules obtained from plants and possess anti-inflammatory properties. Herein we characterize the effect of the flavonoids quercetin, genistein, luteolin, and quercetagetin on LPS-activated transduction mechanism regulation in human gingival fibroblasts (HGF). In this study, we investigated the role of the previously mentioned flavonoids on mitogen-activated protein kinase (MAPK) activation induced by LPS obtained from P. gingivalis. Our results showed that LPS treatment induces activation of extracellular signal related kinase 1/2 (ERK1/2), p38, and c-jun-NH(2)-terminal kinase (JNK). All flavonoids demonstrated an inhibitory effect on MAPK activation, interleukin, 1beta, and cyclooxygenase-2 (COX-2) expression, IL-1beta and prostaglandin E2 (PGE2) synthesis. The most active flavonoid was quercetagetin. Finally we found that the treatment with quercetagetin had no effect on cellular viability or in genetic material integrity.

  7. 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.

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

    PubMed Central

    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

  9. 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.

  10. Anti-inflammatory effects of bamboo salt and sodium fluoride in human gingival fibroblasts--An in vitro study.

    PubMed

    Lee, Hye-Jin; Choi, Choong-Ho

    2015-06-01

    Dental caries preventive agents, such as sodium fluoride (NaF) and bamboo salt (BS), are known to cause cellular growth that is characterized by morphological and gene expression changes. This study was designed to investigate the dual effect of NaF and BS on interleukin (IL)-1β-induced gingival inflammation. Under in vitro experimental conditions, exposure to a subcytotoxic dose of IL-1β enhanced human gingival fibroblast inflammation, as characterized by increased levels of inflammation-associated proteins. A combination of NaF and BS significantly protected fibroblasts from IL-1β-induced cellular deterioration. Exposure to NaF and BS induced the cell growth and no changes in viability were found with the Lactate Dehydrogenase Assay (LDH) assay at the NaF and BS concentration analyzed. Molecular analysis demonstrated that NaF and BS increased resistance to inflammation by reduction of IL-1β, IL-8, and tumor necrosis factor (TNF)-α production. In addition, NaF and BS decreased the expression of IL-1β, IL-8, and TNF-α mRNA in IL-1β-induced human gingival fibroblast cells. The study identifies a new role for NaF and BS in the IL-1β-induced inflammation of gingival fibroblasts and provides a potential target for gingival protection.

  11. 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

  12. 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

  13. Effects of tanshinone VI on the hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts of neonatal rats.

    PubMed

    Maki, Toshiyuki; Kawahara, Yuji; Tanonaka, Kouichi; Yagi, Akira; Takeo, Satoshi

    2002-12-01

    The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [3H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10(-8) M endothelin-1 (ET-1), 10(-6) M phenylephrine (PE), or 10(-8) M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10(-5) M tsh. Treatment with 10(-5) M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [3H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5% FBS or 10(-8) M IGF-1 was inhibited by 10(-5) M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10; - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions. Abbreviations. ANP:atrial natriuretic peptide DMEM:Dulbecco-modified Eagle's medium ET-1:endothelin-1 FB-CM:fibroblast-conditioned medium FBS:fetal bovine serum IGF-1:insulin-like growth factor-1 PE:phenylephrine tsh:tanshinone VI

  14. 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.

  15. In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation.

    PubMed

    Fernandez, Tara L; Van Lonkhuyzen, Derek R; Dawson, Rebecca A; Kimlin, Michael G; Upton, Zee

    2014-01-01

    Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

  16. 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

  17. Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

    SciTech Connect

    McCormick, J.J.; Maher, V.M.

    1985-10-01

    A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B/sub 1/ dichloride, a model for the reactive 2,3-epoxide of aflatoxin B/sub 1/. Exponentially growing cells were exposed to agents and assayed for mutations and cell killing. Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation of normal and repair-deficient cells showed this also to be true for transformation.

  18. 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.

  19. 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

  20. 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.

  1. The effect of chloroquine on the distribution of newly synthesized and old β-hexosaminidase in fibroblasts

    PubMed Central

    Vladutiu, Georgirene D.

    1982-01-01

    Most of the newly synthesized β-N-acetyl-d-glucosaminidase (EC 3.2.1.30; β-hexosaminidase) in normal fibroblast cultures is excreted during 24h incubation with serum-free medium. In this study, this new enzyme only comprises about one-half of the excreted pool as determined by a near total inhibition of [14C]leucine incorporation into the excreted enzyme in the presence of cycloheximide, with only a 46% reduction in enzyme activity. These data indicate that nearly equal fractions of new and old enzyme are normally excreted by fibroblasts. Incubation of normal fibroblast cultures with chloroquine (25 μm) for 24h doubled the amount of extracellular β-hexosaminidase activity from 15% to 37% of total culture activity while reducing the incorporation of [14C]leucine into intra- and extracellular enzyme by 66 and 29% of control, respectively. Therefore, it appears that chloroquine inhibited enzyme synthesis while enhancing the excretion of old as well as newly synthesized enzyme. Chloroquine and cycloheximide together reduced the [14C]leucine incorporation into intracellular enzyme by more than either agent alone, indicating a combined effect on enzyme synthesis and/or degradation. β-Hexosaminidase-deficient fibroblasts that contained endocytosed enzyme spontaneously excreted 10% of their enzyme during 24h incubation with serum-free medium and 18% in the presence of mannose 6-phosphate (2 mm). These results indicated that about one-half of the excreted enzyme still possessed its phosphomannosyl recognition residues and actually re-entered the cells. Chloroquine stimulated the excretion of an addition 15% of the total endocytosed enzyme at 48 and 72h after endocytosis. These data suggest that new, old and endocytosed β-hexosaminidase are all excreted by fibroblasts, that this excretion is enhanced by chloroquine, and that a fraction of the excreted enzyme retains its phosphomannosylated residues needed for re-uptake and transport inside the cells. ImagesFig. 1. PMID

  2. Tirilazad amelioriates extracellular effects of photooxidative stress by sealing the membrane of UVA irradiated human dermal fibroblasts.

    PubMed

    Schneider, Lars Alexander; Dissemond, Joachim; Schwamborn, Edith; Wlaschek, Meinhard; Brenneisen, Peter; Scharffetter-Kochanek, Karin

    2006-01-01

    The evaluation of antioxidant medication might provide further tools to protect the skin against the detrimental effects of photooxidative stress. In this context we have previously shown that the lazaroid tirilazad protects fibroblasts effectively against lipid peroxidation (LPO). Now we investigated whether and how tirilazad also influences two typical stress responses after UVA exposure, i.e. IL-6 and collagenase (MMP-1) release. Fibroblasts pre-incubated with tirilazad at a concentration of 30 microM show significantly less IL-6 in the extracellular medium after UVA exposure. Correspondingly, pre-incubation with tirilazad also significantly diminishes the extracellular MMP-1 protein concentration 24h post-irradiation. These effects observed are due to a membrane stabilisation, as tirilazad neither diminishes IL-6 mRNA production nor intracellular IL-6/MMP-1 protein levels after UVA exposure and thus most likely acts by sealing off the cell, delaying the typical leakage of IL-6 and MMP-1.

  3. Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells.

    PubMed Central

    Golzio, M; Mora, M P; Raynaud, C; Delteil, C; Teissié, J; Rols, M P

    1998-01-01

    Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer. PMID:9635756

  4. 199 EFFECTS OF REPROGRAMMING-CONDITIONED MEDIUM ON ULTRAVIOLET RAY A-DAMAGED HUMAN DERMAL FIBROBLASTS.

    PubMed

    Kang, J; Lee, S G; Kang, J H; Park, S-M; Heo, S Y; Lee, S Y; Kim, S; Lo, E; Ahn, K S; Shim, H

    2016-01-01

    Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P<0.05) among the treatments in the order of Repro-, HDF-, and iPSC-CM. In the same context, apoptosis and senescence were significantly different (P<0.05) in the order of iPSC-, HDF-, and Repro-CM. Interestingly, iPSC-CM did not substantially ameliorate UVA-induced damage, suggesting that the conditions optimized to pluripotent stem cells may not be suitable for the recovery from damage in terminally differentiated cells, such as fibroblasts. The RNA-seq analysis was performed to assess the genome-wide transcriptional profile in the process of recovery. Repro- and HDF-CM were categorized more closely than iPSC-CM in hierarchical cluster analysis. In comparison with iPSC-CM, the up-regulated genes by Repro

  5. Chromosome aberrations in human fibroblasts induced by monoenergetic neutrons. I. Relative biological effectiveness.

    PubMed

    Pandita, T K; Geard, C R

    1996-06-01

    The relative biological effectiveness (RBE) of neutrons for many biological end points varies with neutron energy. To test the hypothesis that the RBE of neutrons varies with respect to their energy for chromosome aberrations in a cell system that does not face interphase death, we studied the yield of chromosome aberrations induced by monoenergetic neutrons in normal human fibroblasts at the first mitosis postirradiation. Monoenergetic neutrons at 0.22, 0.34, 0.43, 1, 5.9 and 13.6 MeV were generated at the Accelerator Facility of the Center for Radiological Research, Columbia University, and were used to irradiate plateau-phase fibroblasts at low absorbed doses from 0.3 to 1.2 Gy at a low dose rate. The reference low-LET, low-dose-rate radiation was 137Cs-gamma rays (0.66 MeV). A linear dose response (Y = alphaD) for chromosome aberrations was obtained for all monoenergetic neutrons and for the gamma rays. The yield of chromosome aberrations per unit dose was high at low neutron energies (0.22, 0.34 and 0.43 MeV) with a gradual decline with the increase in neutron energy. Maximum RBE (RBEm) values varied for the different types of chromosome aberrations. The highest RBE (24.3) for 0.22 and 0.43 MeV neutrons was observed for intrachromosomal deletions, a category of chromosomal change common in solid tumors. Even for the 13.6 MeV neutrons the RBEm (11.1) exceeded 10. These results show that the RBE of neutrons varies with neutron energy and that RBEs are dissimilar between different types of asymmetric chromosome aberrations and suggest that the radiation weighting factors applicable to low-energy neutrons need firmer delineation. This latter may best be attained with neutrons of well-defined energies. This would enable integrations of appropriate quality factors with measured radiation fields, such as those in high-altitude Earth atmosphere. The introduction of commercial flights at high altitude could result in many more individuals being exposed to neutrons than

  6. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization.

    PubMed Central

    Rodrigues, C. M.; Solá, S.; Silva, R.; Brites, D.

    2000-01-01

    BACKGROUND: The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. MATERIALS AND METHODS: Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot. RESULTS: Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05). CONCLUSION: UCB and Abeta peptide

  7. Anti-Inflammatory Effects of TRAF-Interacting Protein in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

    PubMed Central

    Yan, Shi

    2016-01-01

    Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by inflammatory cell infiltration, synovial inflammation, and cartilage destruction. Proliferative fibroblast-like synoviocytes (FLS) play crucial roles in both propagation of inflammation and joint damage because of their production of great amount of proinflammatory cytokines and proteolytic enzymes. In this study, we investigate the role of TRAF-interacting protein (TRIP) in regulating inflammatory process in RA-FLS. TRIP expression was attenuated in RA-FLS compared with osteoarthritis- (OA-) FLS. Overexpression of TRIP significantly inhibited the activation of NF-κB signaling and decreased the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) in TNFα-stimulated RA-FLS. Furthermore, TRIP was found to interact with transforming growth factor β-activated kinase 1 (TAK1) and promoting K48-linked polyubiquitination of TAK1 in RA-FLS. Our results demonstrate that TRIP has anti-inflammatory effects on RA-FLS and suggest TRIP as a potential therapeutic target for human RA. PMID:27847407

  8. Effect of gingival fibroblasts and ultrasound on dogs' root resorption during orthodontic treatment

    PubMed Central

    Crossman, Jacqueline; Hassan, Ali H; Saleem, Ali; Felemban, Nayef; Aldaghreer, Saleh; Fawzi, Elham; Farid, Mamdouh; Abdel-Ghaffar, Khaled; Gargoum, Ausama; El-Bialy, Tarek

    2017-01-01

    Objectives: To investigate the effect of using osteogenic induced gingival fibroblasts (OIGFs) and low intensity pulsed ultrasound (LIPUS) on root resorption lacunae volume and cementum thickness in beagle dogs that received orthodontic tooth movement. Materials and Methods: Seven beagle dogs were used, from which gingival cells (GCs) were obtained and were induced osteogenically to produce OIGFs. Each third and fourth premolar was randomly assigned to one of the five groups, namely, LIPUS, OIGFs, bone morphogenetic protein-2 (BMP-2), OIGFs + LIPUS, and control. All groups received 4 weeks of bodily tooth movement, then LIPUS-treated groups received LIPUS for 20 min/day for 4 weeks, and OIGFs groups received an injection of OIGFs near the root apex. Microcomputed tomography analysis was used to calculate root resorption lacunae volume and histomorphometric analysis was performed to measure the cementum thickness of each root at 3 root levels on compression and tension sides. Results: There was no significant difference in resorption volume between the treatment groups. OIGFs + LIPUS increased cementum thickness (P > 0.05) in third premolars near the apex, and LIPUS increased cementum thickness (P > 0.05) in fourth premolars near the apex. Furthermore, BMP2 increased cementum thickness at the coronal third at the compression side. Conclusion: OIGFs, LIPUS, and BMP-2 can be potential treatments for orthodontically induced root resorption, however, improvements in experimental design and treatment parameters are required to further investigate these repair modalities. PMID:28197400

  9. Effect of trehalose coating on basic fibroblast growth factor release from tailor-made bone implants.

    PubMed

    Choi, Sungjin; Lee, Jongil; Igawa, Kazuyo; Suzuki, Shigeki; Mochizuki, Manabu; Nishimura, Ryohei; Chung, Ung-il; Sasaki, Nobuo

    2011-12-01

    Artificial bone implants are often incorporated with osteoinductive factors to facilitate early bone regeneration. Calcium phosphate, the main component in artificial bone implants, strongly binds these factors, and in a few cases, the incorporated proteins are not released from the implant under conditions of physiological pH, thereby leading to reduction in their osteoinductivity. In this study, we coated tailor-made bone implants with trehalose to facilitate the release of basic fibroblast growth factor (bFGF). In an in vitro study, mouse osteoblastic cells were separately cultured for 48 hr in a medium with a untreated implant (T-), trehalose-coated implant (T+), bFGF-incorporated implant (FT-), and bFGF-incorporated implant with trehalose coating (FT+). In the FT+ group, cell viability was significantly higher than that in the other groups (P<0.05). Scanning electron microscopy (SEM) and X-ray diffraction (XRD) revealed that trehalose effectively covered the surface of the artificial bone implant without affecting the crystallinity or the mechanical strength of the artificial bone implant. These results suggest that coating artificial bone implants with trehalose could limit the binding of bFGF to calcium phosphate.

  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 UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts.

    PubMed

    Hoerter, James D; Ward, Christopher S; Bale, Kyle D; Gizachew, Admasu N; Graham, Rachelle; Reynolds, Jaclyn; Ward, Melanie E; Choi, Chesca; Kagabo, Jean-Leonard; Sauer, Michael; Kuipers, Tara; Hotchkiss, Timothy; Banner, Nate; Chellson, Renee A; Ohaeri, Theresa; Gant, Langston; Vanderhill, Leah

    2008-02-19

    During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.

  12. Protecting effect of phytoncide solution, on normal human dermal fibroblasts against reactive oxygen species.

    PubMed

    Fujimori, Hiroaki; Hisama, Masayoshi; Shibayama, Hiroharu; Iwaki, Masahiro

    2009-01-01

    Four types of phytoncide solutions (A-Type, AB-Type, D-Type and G-Type) was evaluated for reduction of cell damage induced by oxidative stress, ultraviolet A (UVA), ultraviolet B (UVB), hydroxyperoxide (H2O2) and t-butyl-hydroperoxide (t-BHP); stimulation of collagen synthesis against UVA irradiation; and inhibition of matrix metalloproteinase-1 (MMP-1) activity induced by UVA in human normal dermal fibroblasts and human reconstituted skin model. The A-Type, AB-Type, D-Type and G-Type of phytoncide solutions pretreatment resulted in significant protection against cell damage induced by UVB, UVA, H2O2 and t-BHP. The amount of type I collagen following UVA irradiation was increased by treatment with phytoncide solutions in a concentration-dependent manner. On the other hand, phytoncide solutions also suppressed the excess MMP-1 irradiated UVA in a concentration-dependent manner. These effects of G-type solution were superior to those of other types solutions.

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

    PubMed

    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.

  14. UV-protective effects of phytoecdysteroids from Microsorum grossum extracts on human dermal fibroblasts.

    PubMed

    Ho, Raimana; Teai, Taivini; Meybeck, Alain; Raharivelomanana, Phila

    2015-01-01

    Microsorum grossum (Polypodiaceae), locally called metuapua'a, is one of the most frequently used fern species in Polynesian traditional medicine. Fronds or rhizomes of this species are common ingredients of popular medicine recipes to cure various ailments. M. grossum frond and rhizome extracts contain, as their main bioactive components, phytoecdysteroids such as 20-hydroxyecdysone, known to have many interesting biological activities and considered to be adaptogenic compounds [1]. The skin-active effect of M. grossum extract was investigated in two ways on human dermal fibroblasts: a transcriptomic study with c-DNA array for gene expression modulation and a Stress Induced Premature Senescence (SIPS) test. The total extract of M. grossum up-regulates Heme Oxygenase 1 (HO1), an enzyme which protects cells from oxidative stress and which is responsible for skin photoimmunoprotection. The present paper also reports that premature senescence of human skin induced by repeated UV irradiations can be prevented by an ecdysteroid fraction of M. grossum. Our data indicate that extracts of M. grossum could protect skin against oxidative stresses and suggest that they could be used as innovative active cosmetic ingredients.

  15. Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts.

    PubMed Central

    Boegheim, J P; Dubbelman, T M; Mullenders, L H; Van Steveninck, J

    1987-01-01

    Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step. PMID:2965572

  16. 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

  17. Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells

    SciTech Connect

    Chiou, Michael; Xu Yue; Longaker, Michael T. . E-mail: Longaker@stanford.edu

    2006-05-05

    Adipose-derived mesenchymal cells (AMCs) have demonstrated a great capacity for differentiating into bone, cartilage, and fat. Studies using bone marrow-derived mesenchymal cells (BMSCs) have shown that fibroblast growth factor (FGF)-2, a potent mitogenic factor, plays an important role in tissue engineering due to its effects in proliferation and differentiation for mesenchymal cells. The aim of this study was to investigate the function of FGF-2 in AMC chondrogenic differentiation and its possible contributions to cell-based therapeutics in skeletal tissue regeneration. Data demonstrated that FGF-2 significantly promoted the proliferation of AMCs and enhanced chondrogenesis in three-dimensional micromass culture. Moreover, priming AMCs with treatment of FGF-2 at 10 ng/ml demonstrated that cells underwent chondrogenic phenotypic differentiation, possibly by inducing N-Cadherin, FGF-receptor 2, and transcription factor Sox9. Our results indicated that FGF-2 potentiates chondrogenesis in AMCs, similar to its functions in BMSCs, suggesting the versatile potential applications of FGF-2 in skeletal regeneration and cartilage repair.

  18. 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

  19. 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.

  20. Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts.

    PubMed

    Seo, Young-Kwon; Song, Kye-Yong; Kim, Young-Jin; Park, Jung-Keug

    2007-07-01

    In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.

  1. 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.

  2. 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.

  3. 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

  4. Effect of spermine synthase deficiency on polyamine biosynthesis and content in mice and embryonic fibroblasts, and the sensitivity of fibroblasts to 1,3-bis-(2-chloroethyl)-N-nitrosourea.

    PubMed Central

    Mackintosh, C A; Pegg, A E

    2000-01-01

    Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289-295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541-547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1, 3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors

  5. Permeabilization and cell surgery using femtosecond laser pulses: an emerging tool for cellular manipulation

    NASA Astrophysics Data System (ADS)

    Kohli, Vikram; Acker, Jason P.; Elezzabi, Abdulhakem Y.

    2006-02-01

    Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein, we report on the application of femtosecond laser pulses for cellular manipulation, and the generation of optical pores for cytoplasmic delivery of non-reducing cryoprotectants. Under precise laser focusing, we demonstrate membrane surgery on live mammalian cells, and ablation of focal adhesions adjoining fibroblast cells. In both studies, the morphology of the cell post-laser treatment was maintained with no visible collapse or disassociation. When mammalian cells were suspended in a hyperosmotic cryoprotectant solution, focused femtosecond laser pulses were used to transiently permeabilize live cells for sucrose uptake. To verify the cytoplasmic uptake, the volumetric response of cells in 0.2, 0.3, 0.4, and 0.5 M cryoprotective sucrose was measured using video microscopy. From membrane integrity assays, we determined that optimal cell survival of 91.5 +/- 8% is achieved using 0.2 M sucrose, with a decline in survival at higher concentrations. Using diffusion analysis for a porous membrane, the intracellular accumulation of cryoprotective sucrose was theoretically determined. At a diffusion length of 10 um, > 70% of the extracellular osmolarity was estimated to be intracellularly delivered following closure of the transient pore. We anticipate that our study will have important applications for biopresevation, and profound implications for surgery and cell-isolation.

  6. 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.

  7. In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells.

    PubMed

    Martinez, Elizabeth F; Donato, Tatiani A G; Arana-Chavez, Victor E

    2012-10-01

    Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

  8. Ca(2+)-loading modulates potencies of cyclosporin A, Mg2+ and ADP to recouple permeabilized rat liver mitochondria.

    PubMed

    Andreyev AYu; Mikhaylova, L M; Starkov, A A; Kushnareva YuE

    1994-09-01

    We studied the relative potencies of cyclosporin A and endogenous effectors (Mg2+ and ADP) to recouple rat liver mitochondria permeabilized by different Ca(2+)-loading in a P(i)-containing medium. Recoupling efficiency of cyclosporin A dramatically decreased at high Ca(2+)-loading (approx. 100 nM of Ca2+/mg protein and more). Mitochondria permeabilized by high Ca2+ were recoupled with approximately equal efficiency by higher cyclosporin A concentrations or by adding 1-5 mM Mg2+ together with low concentrations of cyclosporin A while potentiating effect of ADP on the cyclosporin A recoupling potency was insignificant. Mg2+ ions at concentrations of 3 mM and higher also prevented the carboxyatractylate-induced reversion of cyclosporin A recoupling effect. The data point to competitive relationships between cyclosporin A and/or Mg2+ ions and Ca2+ ions for the site(s) regulating permeability state of the pore.

  9. Effects of high-energy shockwaves on normal human fibroblasts in suspension.

    PubMed

    Kaulesar Johannes, E J; Sukul, D M; Bijma, A M; Mulder, P G

    1994-12-01

    To gain insight in the effects of shockwaves on human cells the relationship between the energy density and the number of shockwaves as well as their effect on suspensions of normal cells was studied. At energy densities of 0.37, 0.6, 0.78, and 1.20 mJ/mm2 fibroblasts were subjected to 50, 100, 250, 500, and 1,000 shockwaves. Each test was performed three times and one sample was used as control. A decrease in viability related to the logarithm of both the number (P = 0.0000) and the energy density (P = 0.001) of the shockwaves was statistically demonstrable 1 hr after the shockwave application. The energy density of the shockwaves has less influence on the viability than the number of applied shockwaves. Seeding of viable cells 1 hr after the shockwave application showed that the decrease in the 48-hr growth potential was statistically dependent of the number of applied shockwaves only (P = 0.0007). After 24 hr no difference in the 48-hr growth potential could be demonstrated between viable shockwave-treated cells and control cells. The literature as well as our own investigations in vitro and in vivo indicate that shockwaves have a logarithmic dose-dependent destructive effect on cells in suspension, but they also seem to have a dose-dependent stimulating influence on the healing process in damaged tissues. Due to the logarithmic relationship between the viability and both the number and energy density of the applied shockwaves it might be expected that even excessive numbers of high-energy-density shockwaves don't soon lead to total destruction of all cells in the suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin

    SciTech Connect

    Ahnert-Hilger, G.; Bhakdi, S.; Gratzl, M.

    1985-10-15

    The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular YWRb and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

  15. Stimulation of 14-3-3 protein and its isoform on histamine secretion from permeabilized rat peritoneal mast cells.

    PubMed

    Fujii, Toshihiro; Ueeda, Takayuki

    2002-12-01

    The effect of the 14-3-3 protein, an adaptor protein of intracellular signal pathways, on histamine release from rat peritoneal mast cells was investigated. The exogenous 14-3-3 protein from bovine brain increased the Ca(2+)-dependent histamine release from permeabilized mast cells, but only slightly affected the non-permeabilized cells. Partial amino acid sequences showed that the bovine brain 14-3-3 protein contained 14-3-3beta, gamma and zeta isoforms, and that these recombinant isoforms were prepared. Among them, 14-3-3zeta was an active species while the 14-3-3beta and gamma were inactive for histamine release from the permeabilized mast cells. Approximately 15% of the histamine release was stimulated by 14-3-3zeta at 2.5 microM, and half-maximal stimulation occurred at 1 microM. Treatment of the mast cells with wortmannin or staurosporine completely inhibited the stimulatory effect on histamine release caused by Ca(2+) or Ca(2+)/14-3-3zeta, and genistein partially inhibited both stimulatory effects. PD 98059, however, had little effect on the histamine release. These results suggest the possibility that 14-3-3zeta is associated with signal transduction for degranulation of the mast cells.

  16. Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

    PubMed

    Novickij, Vitalij; Lastauskienė, Eglė; Švedienė, Jurgita; Grainys, Audrius; Staigvila, Gediminas; Paškevičius, Algimantas; Girkontaitė, Irutė; Zinkevičienė, Auksė; Markovskaja, Svetlana; Novickij, Jurij

    2017-02-25

    Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 μs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.

  17. 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.

  18. 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.

  19. 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.

  20. Effects of Photodynamic Therapy Using Yellow LED-light with Concomitant Hypocrellin B on Apoptotic Signaling in Keloid Fibroblasts

    PubMed Central

    Hu, Yongqing; Zhang, Chunmin; Li, Shengli; Jiao, Ya; Qi, Tonggang; Wei, Guo; Han, Gangwen

    2017-01-01

    Keloid is a common and refractory disease characterized by abnormal fibroblast proliferation and excessive deposition of extracellular matrix components. Hypocrellin B (HB) is a natural perylene quinone photosensitizer. In this experiment, we studied the effects of photodynamic therapy (PDT) using yellow light from light-emitting diode (LED) combined with HB on keloid fibroblasts (KFB) in vitro. Our results showed that HB-LED PDT treatment induced significant KFB apoptosis and decreased KFB cell viability. HB-LED PDT treatment lead to significant BAX upregulation and BCL-2 downregulation in KFB cells, which led to elevation of intracellular free Ca2+ and activation of caspase-3. Our data provides preliminary evidence for the potential of HB-LED PDT as a therapeutic strategy for keloid. PMID:28367096

  1. Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

    PubMed Central

    Koopmann, W R; Jackson, R C

    1990-01-01

    We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur. Images Fig. 7. PMID:1689146

  2. [Effects of silencing Smad ubiquitination regulatory factor 2 on the function of human hypertrophic scar-derived fibroblasts].

    PubMed

    Zhang, Z; Kuang, F; Liu, C L; Chen, B; Tang, W B; Li, X J

    2017-03-20

    Objective: To explore the effects of silencing Smad ubiquitination regulatory factor 2 (Smurf2) on the secretion of transforming growth factor beta 1 (TGF-β(1)), alpha-smooth muscle actin (α-SMA), and collagen type Ⅰ by human hypertrophic scar-derived fibroblasts. Methods: The human normal skin-derived fibroblasts and hypertrophic scar-derived fibroblasts were cultured with explant culture technique from the normal skin and hypertrophic scar tissue, which was obtained from 9 patients with hypertrophic scars after burn. Two kinds of fibroblasts of the third passage were both divided into 6 groups according to the random number table, with 9 wells in each group. Fibroblasts in blank control group were cultured for 72 h without transfection of any small interfering RNA (siRNA), fibroblasts in negative control group were for cultured for 72 h after transfected with non-target siRNA, fibroblasts in Smurf2 siRNA group were cultured for 72 h after transfected with 100 nmol/L Smurf2 siRNA, fibroblasts in blank control+ TGF-β(1) group were cultured for 72 h without transfection of any siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in negative control+ TGF-β(1) group were cultured for 72 h after transfected with non-target siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h, fibroblasts in Smurf2 siRNA+ TGF-β(1) group were cultured for 72 h after transfected with Smurf2 siRNA and then treated with 10 ng/mL TGF-β(1) for 6 h. (1) The protein and mRNA expression levels of Smurf2 of the two kinds of cells in blank control group, negative control group, and Smurf2 siRNA group were assessed by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively. (2) The content of TGF-β(1) in the cell culture supernatant of the two kinds of cells in blank control group and Smurf2 siRNA group was determined by enzyme-linked immunosorbent assay (ELISA). (3) The protein expression levels of α-SMA of the two kinds of cells in

  3. Effects of 35 kHz, low-frequency ultrasound application in vitro on human fibroblast morphology and migration patterns.

    PubMed

    Conner-Kerr, Teresa; Malpass, Gloria; Steele, Arhalia; Howlett, Allyn

    2015-03-01

    Low-frequency ultrasound (LFU) in the frequency range 30-40 kHz administered using different delivery methods (waterbath and noncontact spray) has shown positive effects on chronic wound healing rates in humans, but the underlying mechanisms are only beginning to be explored. To examine the effects of LFU delivered at 35 kHz on the morphology and migration of human fibroblasts, real-time videography was used to record the rate and character of cultured human fibroblast migration at 8-hour increments for 48 hours in a wound assay model. Cells were treated with 35 kHz LFU or saline only (control). Cellular morphology (cell shape, packing, and apparent length) and migration patterns including orientation were analyzed using time-lapse videography. LFU delivered at a frequency of 35 kHz produced a different pattern of fibroblast migration in vitro (varied orientation versus vertical orientation for untreated cells) and altered cell morphology compared to controls. The observed pattern of migration was more varied and widely distributed across multiple angles versus the predominant parallel orientation of the migrating untreated cells. The potential implications of these findings on collagen placement in the extracellular matrix, which may affect degree of soft tissue scarring, should be further investigated.

  4. Effect of Wubeizi ointment aqueous solution on the expression of type I and III procollagen genes in keloid fibroblasts

    PubMed Central

    Zhai, Xiao-Xiang; Ding, Ji-Cun; Tang, Zhi-Ming; Li, Jing-Guo; Chen, Xiang-Hui; Zhang, Cui-Xia

    2017-01-01

    We evaluated the effect of Wubeizi (WBZ) ointment on keloids. Keloid-derived fibroblast primary cultures were used to evaluate the effect of the different concentration of WBZ ointment on the expression of type I and III procollagen in keloid fibroblast primary cultures using dot blot assay. Type I and II precollagen cDNA probes labeled with non-radioactive digoxin were used for dot blot. Cell cultures were divided into 4 groups: The large dose group received 1 g/ml of WBZ, middle dose, and small dose groups received 0.5 and 0.25 g/ml of WBZ, respectively. The control group received serum-free medium without WBZ. Our results showed that type I and III procollagen mRNA expression was reduced significantly in the large dose and middle dose groups compared to the control group. Type I and III procollagen mRNA expression level in the small dose group had no statistically significant difference with the control group. However, the difference between the large dose group and the small dose group was statistically significant. We concluded that WBZ ointment aqueous solution restricted keloid fibroblast proliferation by downregulating the expression of type I and III procollagen and therefore reducing collagen deposition in keloid tissue.

  5. 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

    2016-04-25

    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.

  6. Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts.

    PubMed Central

    Jordan, N. J.; Watson, M. L.; Yoshimura, T.; Westwick, J.

    1996-01-01

    1. Rheumatoid arthritis is associated with the accumulation and activation of selected populations of inflammatory cells within the arthritic joint. One putative signal for this process is the production, by resident cells, of a group of inflammatory mediators known as the chemokines. 2. The chemokines interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated on activation normal T-cell expressed and presumably secreted) are target-cell specific chemoattractants produced by synovial fibroblasts in response to stimulation with interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha). The signalling pathways involved in their production are not well defined. We therefore used four different protein kinase C inhibitors to investigate the role of this kinase in the regulation of chemokine mRNA and protein expression in human cultured synovial fibroblasts. 3. The non-selective PKC inhibitor, staurosporine (1-300 nM) significantly increased the production of IL-1 alpha-induced IL-8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1-3 microM), also caused a small concentration-dependent increase in IL-8 mRNA and protein production. In contrast, 3-[1-[3-(amidinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl )- 1H-pyrrole-2,5-dione methanesulphonate (Ro 31-8220) and 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3- yl)-maleimide (GF 109203X), two selective PKC inhibitors of the substituted bisindolylmaleimide family had a concentration-dependent biphasic effect on IL-1 alpha or TNF alpha-induced chemokine expression. At low concentrations they caused a stimulation in chemokine production, which was especially evident at the mRNA level. At higher concentrations both inhibited IL-1 alpha or TNF alpha-induced chemokine mRNA and protein production. Ro 31-8220 was 10 fold more potent than GF 109203X, with an IC50 of 1.6 +/- 0.08 microM (mean +/- s.e.mean, n = 4) for IL-1 alpha induced IL-8 production. Ro 31

  7. Effects of oxygen, growth state, and senescence on the antioxidant responses of WI-38 fibroblasts

    PubMed Central

    Reimer, Richard J.; Reenstra, Wende R.; Lilie, Steven M.; Leong, Ina; Sullivan, Katherine; Allen, Robert G.

    2010-01-01

    Mitotically active, growth-arrested cells and proliferatively senescent cultures of human fetal lung fibroblasts (WI-38) were exposed to six different oxygen tensions for various lengths of time and then analyzed to determine the responses of their antioxidant defense system. Glutathione (GSH) concentration increased as a function of ambient oxygen tension in early passage cultures; the effect was larger in exponentially growing cultures than in those in a state of contact-inhibited growth arrest, but was absent in senescent cells. Conversely, the activity of glutathione disulfide reductase was greater in growth-arrested cultures than in mitotically active cells irrespective of oxygen tension. Glucose-6-phosphate dehydrogenase was lowest in log-phase cells exposed to different oxygen tensions for 24 h and in senescent cells. Both hypoxia and hyperoxia depressed selenium-dependent glutathione peroxidase activity in early passage cultures, while the activity of the enzyme progressively declined with increasing oxygen in senescent cells. The GSH S-transferase activity was unresponsive to changes in ambient oxygen tension in either young or senescent cultures. Manganese-containing superoxide dismutase (MnSOD) activity was unaffected by oxygen tension, but was elevated in young confluent cultures as compared with cultures in log-phase growth. MnSOD activity was significantly higher in senescent cultures than in early passage cultures and was also responsive to increased oxygen tension in senescent cultures. Copper–zinc-containing superoxide dismutases activity was not affected by oxygen tension or the passage of time, but it declined in senescent cultures. PMID:20473639

  8. Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis

    PubMed Central

    Li, Yi; Zheng, Jia-Yi; Liu, Jian-Qun; Yang, Jie; Liu, Yang; Wang, Chen; Ma, Xiao-Nan; Liu, Bao-Lin; Xin, Gui-Zhong; Liu, Li-Fang

    2016-01-01

    Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-β (TGF-β1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-β1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-β1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1β further increased TGF-β1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-β1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis. PMID:28003810

  9. 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

  10. 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

  11. 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

    2011-05-11

    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.

  12. [Effect of cetirizine hydrochloride on the expression of substance P receptor and cytokines production in human epidermal keratinocytes and dermal fibroblasts].

    PubMed

    Liu, Ji-Yong; Zhao, Yong-Zhe; Peng, Cheng; Li, Feng-Qian; Zhu, Quan-Gang; Hu, Jin-Hong

    2008-04-01

    To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.

  13. Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

    PubMed Central

    Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Álvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

    2012-01-01

    BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. PMID:22747838

  14. Effect of early administration of exogenous basic fibroblast growth factor on acute edematous pancreatitis in rats

    PubMed Central

    Yan, Qiang; Yao, Xing; Dai, Li-Cheng; Zhang, Guo-Lei; Ping, Jin-Liang; He, Jian-Fang; Han, Chun-Fan

    2006-01-01

    AIM: To observe the therapeutic effect of early administration of exogenous Basic fibroblast growth factor (bFGF) on acute edematous pancreatitis (AEP) in rats. METHODS: Thirty male Sprague-Dawley rats were randomly divided into three (n = 10): normal control group (group I), AEP group (group II) and AEP with bFGF treatment group (group III). AEP was induced by subcutaneous injection of cerulein (5.5 μg/kg and 7.5 μg/kg) at 1 h interval into rats of groups II and III. Three hours after induction of AEP, 100 μg/kg bFGF was administrated intraperitoneally for 1h to group III rats. For test of DNA synthesis in acinar cells, 5-bromo-2’-deoxyuridine (BrdU) labeling solution was intraperitoneally injected into the rats of groups II and III 24 h after bFGF treatment. The changes in serum amylase, lipase, pancreatic tissue wet/dry ratio were detected. RESULTS: In bFGF treatment group, there was a significant decrease in the volume of serum amylase, lipase and the pancreatic wet/dry weight ratio(1383.0 ± 94.6 U/L, 194.0 ± 43.6 U/L, 4.32 ± 0.32) compared to AEP group (3464 ± 223.7 U/L, 456 ±68.7 U/L, 6.89 ± 0.47) (P < 0.01), and no significant difference was found between bFGF treatment and control group (1289 ± 94.0 U/L, 171 ± 23.4 U/L, 4.12 ± 0.26, P > 0.05). The inflammatory changes such as interstitial edema, polymorphonuclear neutrophils (PMNs) and vacuolization were significantly ameliorated compared to AEP group (P < 0.01). A small number of BrdU-labeled nuclei were observed in acinar cells of AEP rats (1.8 ± 0.3 nuclei/microscopic field, n = 10) while diffuse BrdU-labeled nuclei were found in bFGF-treated rats (18.9 ± 1.4 nuclei/microscopic field, n = 10) (P < 0.01). Immunohistochemical study showed increased DNA synthesis in pancreatic acinar cells. CONCLUSION: Early administration of exogenous bFGF has significant therapeutic effect on cerulein-induced acute edematous pancreatitis in rats. Its mechanism is related to the amelioration of inflammation

  15. Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

    SciTech Connect

    Espinoza, B.; Wharton, W.

    1986-08-01

    Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3,5-cyclic monophosphate concentrations.

  16. 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.

  17. 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)

  18. 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.

  19. The Effect of Phototherapy on Cancer Predisposition Genes of Diabetic and Normal Human Skin Fibroblasts

    PubMed Central

    Tangtrakulwanich, Boonsin; Sangkhathat, Surasak

    2017-01-01

    The purpose of this study was to investigate whether LED light at different wavelengths affects the expression profile of 143 cancer predisposition genes in both diabetic and normal human fibroblasts. In this study, both diabetic and normal fibroblast cell lines were cultured and irradiated with red (635 nm), green (520 nm), and blue (465 nm) LED light for 10 minutes at 0.67 J/cm2 each. After that, mRNA from all cell lines was extracted for microarray analysis. We found that green light activates EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, KIT, NF1, BUB1B, FANCD2, EPCAM, FANCD2, NF, DIS3L2, and RET in normal fibroblast cells, while blue and red light can upregulate RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, KIT, FANCD2, BMPR1A, BUB1B, PDE11A, and RET. Therefore, genetic screening before phototherapy treatment may be required. PMID:28386563

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

    PubMed Central

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

    2016-01-01

    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 2 years 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 104 weeks (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 52 wk for C-FGF23 [Beta coefficient (SE) 18.1 (0.04) %, P = 0.04] and from 24 wk 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

  1. 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

  2. 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

  3. TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

    PubMed

    Locci, P; Baroni, T; Lilli, C; Martinese, D; Marinucci, L; Bellocchio, S; Calvitti, M; Becchetti, E

    1999-03-01

    ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

  4. 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

  5. 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.

  6. Mitochondria and cell death: outer membrane permeabilization and beyond.

    PubMed

    Tait, Stephen W G; Green, Douglas R

    2010-09-01

    Mitochondrial outer membrane permeabilization (MOMP) is often required for activation of the caspase proteases that cause apoptotic cell death. Various intermembrane space (IMS) proteins, such as cytochrome c, promote caspase activation following their mitochondrial release. As a consequence, mitochondrial outer membrane integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the B cell lymphoma 2 (BCL-2) protein family. Following MOMP by pro-apoptotic BCL-2-associated X protein (BAX) or BCL-2 antagonist or killer (BAK), additional regulatory mechanisms govern the mitochondrial release of IMS proteins and caspase activity. MOMP typically leads to cell death irrespective of caspase activity by causing a progressive decline in mitochondrial function, although cells can survive this under certain circumstances, which may have pathophysiological consequences.

  7. 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.

  8. 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

  9. Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

    PubMed

    Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

    2013-02-01

    Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.

  10. 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.

  11. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages.

    PubMed

    Ghosh, Moumita; Carlsson, Fredrik; Laskar, Amit; Yuan, Xi-Ming; Li, Wei

    2011-02-18

    Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH(4)Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.

  12. Genome-wide microarray analysis of human fibroblasts in response to γ radiation and the radiation-induced bystander effect.

    PubMed

    Kalanxhi, Erta; Dahle, Jostein

    2012-01-01

    Radiation-induced bystander effects have been studied extensively due to their potential implications for cancer therapy and radiation protection; however, a complete understanding of the molecular mechanisms remains to be elucidated. In this study, we monitored transcriptional responses to γ radiation in irradiated and bystander fibroblasts simultaneously employing a genome-wide microarray approach to determine factors that may be modulated in the generation or propagation of the bystander effect. For the microarray data we employed analysis at both the single-gene and gene-set level to place the findings in a biological context. Unirradiated bystander fibroblasts that were recipients of growth medium harvested from irradiated cultures 2 h after exposure to 2 Gy displayed transient enrichment in gene sets belonging to ribosome, oxidative phosphorylation and neurodegenerative disease pathways associated with mitochondrial dysfunctions. The response to direct irradiation was characterized by induction of signaling and apoptosis genes and the gradual formation of a cellular immune response. A set of 14 genes, many of which were regulated by p53, were found to be induced early after irradiation (prior to medium transfer) and may be important in the generation or propagation of the bystander effect.

  13. Effects of methylglyoxal on human cardiac fibroblast: roles of transient receptor potential ankyrin 1 (TRPA1) channels.

    PubMed

    Oguri, Gaku; Nakajima, Toshiaki; Yamamoto, Yumiko; Takano, Nami; Tanaka, Tomofumi; Kikuchi, Hironobu; Morita, Toshihiro; Nakamura, Fumitaka; Yamasoba, Tatsuya; Komuro, Issei

    2014-11-01

    Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca(2+) signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca(2+) concentration [Ca(2+)]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca(2+) entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031, a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca(2+) entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca(2+) entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca(2+) entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

  14. Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

    PubMed

    Paolella, Gaetana; Lepretti, Marilena; Barone, Maria Vittoria; Nanayakkara, Merlin; Di Zenzo, Marina; Sblattero, Daniele; Auricchio, Salvatore; Esposito, Carla; Caputo, Ivana

    2017-03-01

    Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

  15. Effects of nanometric roughness on surface properties and fibroblast's initial cytocompatibilities of Ti6Al4V.

    PubMed

    Wang, Rex C-C; Hsieh, Ming-Che; Lee, Tzer-Min

    2011-09-01

    Titanium alloy (Ti6Al4V) has widespread medical applications because of its excellent biocompatibility. Its biological responses can further be enhanced by polishing and passivation. Unfortunately, preparing titanium alloy samples of nanometric roughness is by far much more difficult than preparing those of micrometric roughness, and numerous investigations on roughness induced effects are all focused on micrometric scales. For the remedy, we investigate, at nanometric scale, the influence of roughness on surface properties and biological responses. Six groups of Ti6Al4V with average roughness (R(a)) values of 2.75-30.34 nm are prepared. The results indicated that nanometric roughness of samples change the wettability and amphoteric OH groups. The contact angles monotonically decrease from 2.75 to 30.34 nm and the rougher surfaces lead to higher wettability. The in vitro cell-culture studies, using Murine NIH-3T3 fibroblasts, showed the spindle-shaped morphology on rougher surface compared to round∕spherical morphology on smoother surface. A cytodetacher is employed to quantitatively measure the initial adhesion force of fibroblasts to specimen. The adhesion strength of fibroblasts, ranging from 55 to 193 nN, is significantly influenced by the nanometric roughness while the surface is within the range of 2.75-30.34 nm R(a) roughness, and the adhesion strength appeared stronger for rougher surface. The cell number on the smoother surface is higher than on the rougher surface at 5-day culture. The studies indicated that nanometric roughness would alter the surface properties and further influence cell morphology, adhesion strength, and proliferation.

  16. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts

    PubMed Central

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  17. Effects of cigarette smoke residues from textiles on fibroblasts, neurocytes and zebrafish embryos and nicotine permeation through human skin.

    PubMed

    Hammer, Timo R; Fischer, Kirsten; Mueller, Marina; Hoefer, Dirk

    2011-09-01

    Toxic substances from cigarette smoke can attach to carpets, curtains, clothes or other surfaces and thus may pose risks to affected persons. The phenomenon itself and the potential hazards are discussed controversially, but scientific data are rare. The objective of this study was to examine the potential of textile-bound nicotine for permeation through human skin and to assess the effects of cigarette smoke extracts from clothes on fibroblasts, neurocytes and zebrafish embryos. Tritiated nicotine from contaminated cotton textiles penetrated through adult human full-thickness skin as well as through a 3D in vitro skin model in diffusion chambers. We also observed a significant concentration-dependent cytotoxicity of textile smoke extracts on fibroblast viability and structure as well as on neurocytes. Early larval tests with zebrafish embryos were used as a valid assay for testing acute vertebrate toxicity. Zebrafish development was delayed and most of the embryos died when exposed to smoke extracts from textiles. Our data show that textiles contaminated with cigarette smoke represent a potential source of nicotine uptake and can provoke adverse health effects.

  18. Effect of myocyte-fibroblast coupling on the onset of pathological dynamics in a model of ventricular tissue

    NASA Astrophysics Data System (ADS)

    Sridhar, S.; Vandersickel, Nele; Panfilov, Alexander V.

    2017-01-01

    Managing lethal cardiac arrhythmias is one of the biggest challenges in modern cardiology, and hence it is very important to understand the factors underlying such arrhythmias. While early afterdepolarizations (EAD) of cardiac cells is known to be one such arrhythmogenic factor, the mechanisms underlying the emergence of tissue level arrhythmias from cellular level EADs is not fully understood. Another known arrhythmogenic condition is fibrosis of cardiac tissue that occurs both due to aging and in many types of heart diseases. In this paper we describe the results of a systematic in-silico study, using the TNNP model of human cardiac cells and MacCannell model for (myo)fibroblasts, on the possible effects of diffuse fibrosis on arrhythmias occurring via EADs. We find that depending on the resting potential of fibroblasts (VFR), M-F coupling can either increase or decrease the region of parameters showing EADs. Fibrosis increases the probability of occurrence of arrhythmias after a single focal stimulation and this effect increases with the strength of the M-F coupling. While in our simulations, arrhythmias occur due to fibrosis induced ectopic activity, we do not observe any specific fibrotic pattern that promotes the occurrence of these ectopic sources.

  19. Effect of myocyte-fibroblast coupling on the onset of pathological dynamics in a model of ventricular tissue

    PubMed Central

    Sridhar, S.; Vandersickel, Nele; Panfilov, Alexander V.

    2017-01-01

    Managing lethal cardiac arrhythmias is one of the biggest challenges in modern cardiology, and hence it is very important to understand the factors underlying such arrhythmias. While early afterdepolarizations (EAD) of cardiac cells is known to be one such arrhythmogenic factor, the mechanisms underlying the emergence of tissue level arrhythmias from cellular level EADs is not fully understood. Another known arrhythmogenic condition is fibrosis of cardiac tissue that occurs both due to aging and in many types of heart diseases. In this paper we describe the results of a systematic in-silico study, using the TNNP model of human cardiac cells and MacCannell model for (myo)fibroblasts, on the possible effects of diffuse fibrosis on arrhythmias occurring via EADs. We find that depending on the resting potential of fibroblasts (VFR), M-F coupling can either increase or decrease the region of parameters showing EADs. Fibrosis increases the probability of occurrence of arrhythmias after a single focal stimulation and this effect increases with the strength of the M-F coupling. While in our simulations, arrhythmias occur due to fibrosis induced ectopic activity, we do not observe any specific fibrotic pattern that promotes the occurrence of these ectopic sources. PMID:28106124

  20. The safety and effect of topically applied recombinant basic fibroblast growth factor on the healing of chronic pressure sores.

    PubMed Central

    Robson, M C; Phillips, L G; Lawrence, W T; Bishop, J B; Youngerman, J S; Hayward, P G; Broemeling, L D; Heggers, J P

    1992-01-01

    The first randomized, blinded, placebo-controlled human trials of recombinant basic fibroblast growth factor (bFGF) for pressure sore treatment were performed. Three different concentrations of bFGF in five dosing schedules were tested for safety using hematology, serum chemistries, urinalysis, absorption, antibody formation, and signs of toxicity. Efficacy was evaluated by wound volumes, histology, and photography. No toxicity, significant serum absorption, or antibody formation occurred. In six of eight subgroups, there was a trend toward efficacy with bFGF treatment. When all subgroups were combined, comparison of the slopes of the regression curves of volume decrease over initial pressure sore volume demonstrated a greater healing effect for the bFGF-treated patients (p < 0.05). Histologically, bFGF-treated wound sections demonstrated increased fibroblasts and capillaries. More patients treated with bFGF achieved > 70% wound closure (p < 0.05). Blinded observers were able to distinguish differences in visual wound improvement between bFGF and placebo groups. These data suggest that bFGF may be effective in the treatment of chronic wounds. PMID:1417189

  1. Sodium alginate and gum acacia hydrogels of ZnO nanoparticles show wound healing effect on fibroblast cells.

    PubMed

    Raguvaran, R; Manuja, Balvinder K; Chopra, Meenu; Thakur, Rajesh; Anand, Taruna; Kalia, Anu; Manuja, Anju

    2017-03-01

    An ideal biomaterial for wound dressing applications should possess antibacterial and anti-inflammatory properties without any toxicity to the host cells while providing the maximum healing activity. Zinc oxide nanoparticles (ZnONPs) possess antimicrobial activity and enhance wound healing, but the questions regarding their safety arise before application to the biological systems. We synthesized ZnONPs-loaded-sodium alginate-gum acacia hydrogels (SAGA-ZnONPs) by cross linking hydroxyl groups of the polymers sodium alginate and gum acacia with the aldehyde group of gluteradehyde. Here, we report the wound healing properties of sodium alginate/gum acacia/ZnONPs, circumventing the toxicity of ZnONPs simultaneously. We demonstrated the concentration-dependent zones of inhibition in treated cultures of Pseudomonas aerigunosa and Bacillus cereus and biocompatability on peripheral blood mononuclear/fibroblast cells. SAGA-ZnONPs hydrogels showed a healing effect at a low concentration of ZnONPs using sheep fibroblast cells. Our findings suggest that high concentrations of ZnONPs were toxic to cells but SAGA-ZnONPs hydrogels significantly reduced the toxicity and preserved the beneficial antibacterial and healing effect.

  2. Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons.

    PubMed

    Matalonga, Leslie; Arias, Ángela; Tort, Frederic; Ferrer-Cortés, Xènia; Garcia-Villoria, Judit; Coll, Maria Josep; Gort, Laura; Ribes, Antonia

    2015-10-01

    Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthrough-promoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.

  3. 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

  4. Analysis of propolis from Baccharis dracunculifolia DC. (Compositae) and its effects on mouse fibroblasts.

    PubMed

    de Funari, Cristiano Soleo; de Oliveira Ferro, Vicente; Mathor, Monica Beatriz

    2007-05-04

    This paper confirms Baccharis dracunculifolia DC. (Compositae) as the main botanical source of the propolis from southeastern Brazil (state of São Paulo) investigated to ascertain specific biological activity in relation to mouse NIH-3T3 fibroblasts, skin cells directly involved in the cicatrization processes. Flavonoid and total phenolic compounds were determined by spectrophotometry, and chemical composition by HPLC; the chromatographic profile, characterized largely by flavonoids and aromatic acids, was found to be qualitatively similar to that of Baccharis dracunculifolia DC. The adsorption of phenolic compounds in the propolis to skin powder was also investigated, and 68% of these compounds adsorbed to the skin powder. At concentrations from 0.12 to 7.81 microg/ml, the propolis revealed no statistical significant differences from its control solutions; however, at concentrations of 31.25 microg/ml or more, the propolis was toxic to NIH-3T3 cells. Thus, the propolis from Baccharis dracunculifolia DC. (Compositae) presents an in vitro concentration-dependent toxicity on mouse NIH-3T3 fibroblasts.

  5. Polymer chain length effects on fibroblast attachment on nylon-3-modified surfaces.

    PubMed

    Liu, Runhui; Masters, Kristyn S; Gellman, Samuel H

    2012-04-09

    Nylon-3 polymers have a polyamide backbone reminiscent of that found in proteins (β- vs α-amino acid residues, respectively), which makes these materials interesting for biological applications. Because of the versatility of the ring-opening polymerization process and the variety of β-lactam starting materials available, the structure of nylon-3 copolymers is highly amenable to alteration. A previous study showed that relatively subtle changes in the structure or ratio of hydrophobic and cationic subunits that comprise these polymers can result in significant changes in the ability of nylon-3-bearing surfaces to support cell adhesion and spreading. In the present study, we have exploited the highly tailorable nature of these polymers to synthesize new versions possessing a wide range of chain lengths, with the intent of optimizing these materials for use as cell-supportive substrates. We find that longer nylon-3 chains lead to better fibroblast attachment on modified surfaces and that at the optimal chain lengths less hydrophobic subunits are superior. The best polymers we identified are comparable to an RGD-containing peptide in supporting fibroblast attachment. The results described here will help to focus future efforts aimed at refining nylon-3 copolymer substrates for specific tissue engineering applications.

  6. 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.

  7. Mitochondrial respiratory chain inhibitors modulate the metal-induced inner mitochondrial membrane permeabilization.

    PubMed

    Belyaeva, Elena A

    2010-01-01

    To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd²(+), Hg²(+), Cu²(+) and Zn²(+), as well as by Ca²(+) (in the presence of P(i)) or Se (in form of Na₂SeO₃) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascorbate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu²(+)-induced swelling. In contrast, the high-amplitude swelling produced by Cd²(+), Hg²(+), Zn²(+), or Ca²(+) plus P(i) was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.

  8. Effect of soluble products from lectin-stimulated lymphocytes on the growth, adhesiveness, and glycosaminoglycan synthesis of cultured synovial fibroblastic cells.

    PubMed Central

    Anastassiades, T P; Wood, A

    1981-01-01

    Human blood mononuclear cells exposed to concanavalin A or phytohemagglutinin secrete a soluble factor that arrests the growth of human synovial fibroblastic cells in culture. Once the growth-inhibitory effect is initiated it cannot be reversed by washing the fibroblastic cells, by refeeding with nonconditioned fresh serum-containing medium, by trypsinization, EDTA treatment, or a combination of these procedures. Media from nonstimulated mononuclear cells, fibroblastic cells, or the lectins themselves do not contain similar inhibitory activity that can be detected by the present culture systems. This lectin-dependent, growth-inhibitory activity does not have a cytotoxic effect on the fibroblasts but increases their adhesiveness to plastic or glass surfaces, and the cells tend to assume a less fibroblastic morphology. The growth-inhibitory activity is stable in the cold and is nondialyzable or ultrafilterable, but the activity is rapidly lost at temperature between 60 degrees and 70 degrees C and at pH 2.0. The growth-arrested cells secrete more glycosaminoglycan per cell in the medium and synthesize more cell surface glycosaminoglycan than the controls. However, the increased glycosaminoglycan synthesis cannot be explained as being entirely secondary to a cell density effect as it is also observed when adjustments are made for the differences in growth rates. PMID:7276172

  9. Prostacyclin analogs inhibit fibroblast migration.

    PubMed

    Kohyama, Tadashi; Liu, Xiangde; Kim, Hui Jung; Kobayashi, Tetsu; Ertl, Ronald F; Wen, Fu-Qiang; Takizawa, Hajime; Rennard, Stephen I

    2002-08-01

    The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI(2)) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI(2) on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI(2) analog carbaprostacyclin (10(-6) M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 microg/ml) 58.0 +/- 13.2% (P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 +/- 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI(2) analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10(-6) M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI(2) appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.

  10. Saliva improves Streptococcus mitis protective effect on human gingival fibroblasts in presence of 2-hydroxyethyl-methacrylate.

    PubMed

    Di Giulio, Mara; di Giacomo, Viviana; Di Campli, Emanuela; Di Bartolomeo, Soraya; Zara, Susi; Pasquantonio, Guido; Cataldi, Amelia; Cellini, Luigina

    2013-08-01

    This study aimed to investigate the effect of saliva on Streptococcus mitis free cells and on S. mitis/human gingival fibroblasts (HGFs) co-culture model, in presence of 2-hydroxyethyl-methacrylate (HEMA). The bacterial aggregation both in the planktonic phase and on HGFs, as well as the apoptotic and necrotic eukaryotic cells amount were analyzed, in presence of saliva and/or HEMA. The aggregation test revealed a significant saliva aggregation effect on S. mitis strains compared to the untreated sample. No significant differences were recorded in the amount of culturable bacteria in all studied conditions; however, from microscopy images, the saliva/HEMA combining effect induced a significant bacterial aggregation and adhesion on HGFs. HEMA treatment decreased viable eukaryotic cell number with a parallel increment of necrotic cells, but when saliva was added to the co-culture, the viable cells percentage increased to a value comparable to the control sample.

  11. Effects of fibroblast growth factor 2 on osteoblastic proliferation and differentiation by regulating bone morphogenetic protein receptor expression.

    PubMed

    Park, Jun-Beom

    2011-09-01

    Fibroblast growth factors (FGFs) are known to play a critical role in bone growth and development, affecting both osteogenesis and chondrogenesis. Fibroblast growth factor 2 (FGF-2) is produced intracellularly by osteoblasts and secreted into the surrounding matrix in bone.The dose-dependent effects of FGF-2 were tested to examine the relationship between FGF-2 and osteoblast proliferation and differentiation. Tests used included a cell viability test, an alkaline phosphatase activity test, and a Western blot analysis.Cultures growing in the presence of FGF-2 showed an increased value for 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and a decreased value for alkaline phosphatase activity. Results of the Western blot analysis showed that the addition of FGF-2 seems to decrease osteocalcin and bone morphogenetic protein receptor IA.These data show that FGF-2 in the tested dosage within MC3T3-E1 cells seems to affect proliferation and differentiation. Results of the Western blot analysis may add some possible mechanisms, and it may be suggested that treatment of FGF-2 may have an influence on the expression of bone morphogenetic protein receptors in osteoprecursor cells. Further elucidation of the mechanisms related to this mechanism within the in vivo model may be necessary to ascertain greater detail.

  12. 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.

  13. Evaluation of genotoxic effects in human fibroblasts after intermittent exposure to 50 Hz electromagnetic fields: a confirmatory study.

    PubMed

    Scarfí, Maria Rosaria; Sannino, Anna; Perrotta, Alessandro; Sarti, Maurizio; Mesirca, Pietro; Bersani, Ferdinando

    2005-09-01

    The aim of this investigation was to confirm the main results reported in recent studies on the induction of genotoxic effects in human fibroblasts exposed to 50 Hz intermittent (5 min field on/10 min field off) sinusoidal electromagnetic fields. For this purpose, the induction of DNA single-strand breaks was evaluated by applying the alkaline single-cell gel electrophoresis (SCGE)/comet assay. To extend the study and validate the results, in the same experimental conditions, the potential genotoxicity was also tested by exposing the cells to a 50 Hz powerline signal (50 Hz frequency plus its harmonics). The cytokinesis-block micronucleus assay was applied after 24 h intermittent exposure to both sinusoidal and powerline signals to obtain information on cell cycle kinetics. The experiments were carried out on human diploid fibroblasts (ES-1). For each experimental run, exposed and sham-exposed samples were set up; positive controls were also provided by treating cells with hydrogen peroxide or mitomycin C for the comet or micronucleus assay, respectively. No statistically significant difference was detected in exposed compared to sham-exposed samples in any of the experimental conditions tested (P > 0.05). In contrast, the positive controls showed a statistically significant increase in DNA damage in all cases, as expected. Accordingly, our findings do not confirm the results reported previously for either comet induction or an increase in micronucleus frequency.

  14. 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

  15. Effect of Immobilized Thiolated Glycosaminoglycans on Fibronectin Adsorption and Behavior of Fibroblasts.

    PubMed

    Köwitsch, Alexander; Niepel, Marcus S; Michanetzis, Georgios P A; Missirlis, Yannis F; Groth, Thomas

    2016-03-01

    Glycosaminoglycans (GAGs) chondroitin sulfate, heparin, hyaluronan, and sulfated hyaluronan are lower and higher thiolated to enable a one-step covalent modification of gold or vinyl-terminated surfaces. Measurements of water contact angle and zeta potentials reveal that sulfated GAG-modified surfaces are more wettable and possess a negative surface potential. Additionally, higher thiolated GAGs (tGAGs) exhibit increased wettability and higher surface roughness. Fibronectin (FN) adsorption increases with sulfation degree of tGAGs. The tGAG-functionalized surfaces with higher degree of sulfation promote fibroblast adhesion most under serum-free conditions. The preadsorption of FN allows for more cell adhesion on tGAG surfaces. Metabolic activity measurements show that cell growth is enhanced for tGAGs up to a certain thiolation degree. Overall, thiolation of GAGs does not hamper their bioactivity toward proteins and cells, which make them highly interesting for biomimetic surface modification of implants and tissue engineering scaffolds.

  16. Effects of cathepsin D inhibitor from Vicia sativa L. seed hulls on human skin fibroblasts and breast cancer cells (in vitro studies).

    PubMed

    Roszkowska-Jakimiec, W; Wołczyński, S; Chlabicz, M

    2004-01-01

    Cathepsin D is a lysosomal protease which plays an important role in cancer invasion and metastasis. There are known inhibitors of that enzyme, such as pepstatin and potato inhibitor. In this study, we examined effects of the cathepsin D inhibitor from Vicia sativa L. seed hulls on cell cultures of human skin fibroblasts and breast cancer cells. There is no effect of the D-cathepsin inhibitor from Vicia sativa L. seed hulls on the proliferative activity of either human skin fibroblasts or breast cancer cells, measured by the [3H] thymidine incorporation assay.

  17. 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.

  18. Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons.

    PubMed

    Fried, Kaj; Sime, Wondossen; Lillesaar, Christina; Virtanen, Ismo; Tryggvasson, Karl; Patarroyo, Manuel

    2005-07-15

    The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.

  19. Hydrogen peroxide modulates Ca2+-activation of single permeabilized fibres from fast- and slow-twitch skeletal muscles of rats.

    PubMed

    Plant, D R; Lynch, G S; Williams, D A

    2000-01-01

    We examined the effects of redox modulation on single membrane-permeabilized fibre segments from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of adult rats to determine whether the contractile apparatus was the redox target responsible for the increased contractility of muscles exposed to low concentrations of H2O2. The effects of H2O2 on maximum Ca2+-activated force were dose-dependent with 30 min exposure to 5 mM H2O2 causing a progressive decrease by 22+/-3 and 13+/-2% in soleus and EDL permeabilized muscle fibres, respectively. Lower concentrations of exogenous H2O2 (100 microM and 1 mM) had no effect on maximum Ca2+-activated force. Subsequent exposure to the reductant dithiothreitol (DTT, 10 mM, 10 min) fully reversed the H2O2-induced depression of force in EDL, but not in soleus muscle fibres. Incubation with DTT alone for 10 min did not alter Ca2+-activated force in either soleus or EDL muscle fibres. The sensitivity of the contractile filaments to Ca2+ (pCa50) was not altered by exposure to any concentration of exogenous H2O2. However, all concentrations of H2O2 diminished the Hill coefficient in permeabilized fibres from the EDL muscle, indicating that the cooperativity of Ca2+ binding to troponin is altered. H2O2 (5 mM) did not affect rigor force, which indicates that the number of crossbridges participating in contraction was not reduced. In conclusion, H2O2 may reduce the maximum Ca2+ activated force production in skinned muscle fibres by decreasing the force per crossbridge.

  20. Effect of 4-Allyl-1-hydroxy-2-methoxybenzene (Eugenol) on Inflammatory and Apoptosis Processes in Dental Pulp Fibroblasts

    PubMed Central

    Martínez-Herrera, Andrea; Ruiz-Rodríguez, Socorro; Vértiz-Hernández, Antonio

    2016-01-01

    Eugenol (mixed with zinc oxide powder) is widely used as direct capping material during pulp therapy in primary teeth. The aim of the present study was to evaluate the effect of eugenol on diverse genes involved in inflammatory and cell apoptosis processes. The regulatory effect of eugenol on the expression of inflammation and apoptotic genes was evaluated in dental pulp fibroblasts from extracted third molars, cultured under concentration of eugenol of 13 μM. Eugenol allowed the expression of inflammatory and apoptotic genes when compared with positive and negative controls. Eugenol is a proinflammatory agent when it is in direct contact with healthy tissues and behaves as an anti-inflammatory agent in tissues undergoing inflammatory/apoptotic processes, as in cases of pulp inflammation in primary teeth. These findings are relevant for dentistry, when considering the application of safer pulp treatments to grossly carious children's teeth. PMID:28044068

  1. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  2. The effects of macrophage-stimulating protein on the migration, proliferation, and collagen synthesis of skin fibroblasts in vitro and in vivo.

    PubMed

    Zhao, Jiajia; Hu, Li; Gong, Niya; Tang, Qingming; Du, Luyang; Chen, Lili

    2015-03-01

    Macrophage-stimulating protein (MSP), an important cytokine with multiple functions, is highly expressed in adipose-derived stem cells-conditioned medium (ASC-CM). ASCs can effectively promote wound healing through paracrine mechanism, suggesting that MSP may play a critical role in wound healing. Through binding to its receptor, RON (Receptuerd'OrigineNantaise, also called macrophage stimulation 1 receptor; MST1R), it can activate epithelial cells and work as an inflammatory mediator. In this study, we found RON was also expressed on dermal fibroblasts and investigated the effects of MSP on proliferation, migration, and collagen synthesis of fibroblasts. With the treatment of different concentrations of MSP (0, 1, 10, 20, 50, and 100 ng/mL) on fibroblasts, proliferation, migration, and collagen synthesis were analyzed by Cell Counting Kit-8 (CCK-8), transwell and real-time polymerase chain reaction. Under the treatment of MSP, the migration, Collagen I, III synthesis, and matrix metalloproteinase-1 (MMP-1) mRNA expression of fibroblasts were upregulated significantly, although there was no effect on fibroblasts proliferation, and the optimal concentration of MSP for migration and collagen synthesis was 10 ng/mL. In the in vivo study, 10 ng/mL MSP was applied to full-thickness skin wound with bacterial cellulose membranes, and this treatment could accelerate the wound healing rate and increased the collagen synthesis of wound sites. This study suggested that MSP appears to promote the migration of fibroblasts, enhances collagen synthesis and remodeling, and effectively improves wound healing.

  3. 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

  4. The Flavonoid 7,4'-Dihydroxyflavone Prevents Dexamethasone Paradoxical Adverse Effect on Eotaxin Production by Human Fibroblasts.

    PubMed

    Liu, Changda; Yang, Nan; Chen, Xiaoke; Tversky, Jody; Zhan, Jixun; Chehade, Mirna; Miller, Rachel L; Li, Xiu-Min

    2017-03-01

    Eotaxin/CCL-11 is a major chemoattractant that contributes to eosinophilic inflammation in asthma. Glucocorticoids inhibit inflammation, but long-time exposure may cause paradoxical adverse effects by augmenting eotaxin/CCL-11production. The aim of this study was to determine if 7,4'-dihydroxyflavone (7,4'-DHF), the eotaxin/CCL11 inhibitor isolated from Glycyrrhiza uralensis, reduces in vitro eotaxin production induced by long-time dexamethasone (Dex) exposure, and if so, to elucidate the mechanisms of this inhibition. Human lung fibroblast-1 cells were used to identify the potency of 7,4'-DHF compared with other compounds from G. uralensis, to compare 7,4'-DHF with Dex on eotaxin production following 24-h short-time culture and 72-h longer-time (LT) culture, and to determine the effects of the 7,4'-DHF on Dex LT culture augmented eotaxin production and molecule mechanisms. 7,4'-DHF was the most potent eotaxin/CCL-11 inhibitor among the ten compounds and provided continued suppression. In contrast to short-time culture, Dex LT culture increased constitutively, and IL-4/TNF-α stimulated eotaxin/CCL11 production by human lung fibroblast-1 cells. This adverse effect was abrogated by 7,4'-DHF co-culture. 7,4'-DHF significantly inhibited Dex LT culture augmentation of p-STAT6 and impaired HDAC2 expression. This study demonstrated that 7,4'-DHF has the ability to consistently suppress eotaxin production and prevent Dex-paradoxical adverse effects on eotaxin production. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Permeabilization and in situ adsorption studies during growth and coumarin production in hairy root cultures of Cichorium intybus L.

    PubMed

    Bais, H P; Sudha, G; Suresh, B; Ravishankar, G A

    2001-06-01

    Effect of addition of a permeabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied. Continuous permeabilization of the hairy root cultures of C. intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2% (v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % v/v) to hairy root cultures of C. intybus, showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1% media filtrate (MF) of cultures of Phytopthora parasitica var. nicotiana treatment. It was observed that treatment with DMSO (0.2% v/v) and 1% MF of P. parasitica showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD-7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD-7 along with 1% MF of P. parasitica showed enhanced growth, coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO/XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO/XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. intybus by using DMSO at 0.2% (v/v) level coupled with 1% MF of P. parasitica maintained viability of tissues and produced coumarins at higher level.

  6. Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin*

    PubMed Central

    Zhang, TianHua; Muraih, Jawad K.; Tishbi, Nasim; Herskowitz, Jennifer; Victor, Rachel L.; Silverman, Jared; Uwumarenogie, Stephanie; Taylor, Scott D.; Palmer, Michael; Mintzer, Evan

    2014-01-01

    Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic. PMID:24616102

  7. Comparative effects of posterior eye cup tissues from myopic and hyperopic chick eyes on cultured scleral fibroblasts.

    PubMed

    Christian, Parul G; Harkin, Damien G; Rayner, Cassie; Schmid, Katrina L

    2013-02-01

    The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5-8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA

  8. EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION.

    PubMed

    Merlavsky, V M; Manko, B O; Ikkert, O V; Manko, V V

    2015-01-01

    To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and a-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml--by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml--by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate, on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 αg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

  9. 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.

  10. Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro

    PubMed Central

    Wang, Xian-wei; Zhang, Fen-xi; Yang, Fen; Ding, Zu-feng; Agarwal, Nidhi; Guo, Zhi-kun; Mehta, Jawahar L

    2016-01-01

    Aim: Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors can not only lower blood glucose levels, but also alleviate cardiac remodeling after myocardial ischemia and hypertension. In the present study, we investigated the effects of a DPP-4 inhibitor (linagliptin) and a GLP-1 activator (liraglutide) on glucose- and angiotensin II (Ang II)-induced collagen formation and cytoskeleton reorganization in cardiac fibroblasts in vitro, and elucidated the related mechanisms. Methods: Cardiac fibroblasts were isolated from the hearts of 6-week-old C57BL/6 mice, and then exposed to different concentrations of glucose or Ang II for 24 h. The expression of fibrotic signals (fibronectin, collagen-1, -3 and -4), as well as ERK1/2 and NF-κB-p65 in the fibroblasts was examined using Western blotting assays. F-actin degradation was detected under inverted laser confocal microscope in fibroblasts stained with Rhodamine phalloidin. Results: Glucose (1–40 mmol/L) and Ang II (10−8–10−5 mol/L) dose-dependently increased the expression of fibronectin, collagens, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. High concentrations of glucose (≥40 mmol/L) and Ang II (≥10−6 mol/L) caused a significant degradation of F-actin (less assembly F-actin fibers and more disassembly fibers). ERK1/2 inhibitor U0126 (10 μmol/L) and NF-κB inhibitor JSH-23 (10 μmol/L) both markedly suppressed glucose- and angiotensin II-induced fibronectin and collagen expressions in cardiac fibroblasts. Furthermore, pretreatment with liraglutide (10–100 nmol/L) or linagliptin (3 and 30 nmol/L) significantly decreased glucose- and Ang II-induced expression of fibrotic signals, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. Moreover, pretreatment with liraglutide (30 nmol/L) or liraglutide (100 nmol/L) markedly inhibited glucose-induced F-actin degradation, however, only liraglutide inhibited Ang II-induced F-actin degradation. Conclusion

  11. 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

  12. 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.

  13. Inhibitory effects of antioxidant constituents from Melothria heterophylla on matrix metalloproteinase-1 expression in UVA-irradiated human dermal fibroblasts.

    PubMed

    Cho, Y H; Kim, J H; Sim, G S; Lee, B C; Pyo, H B; Park, H D

    2006-01-01

    Matrix metalloproteinases (MMPs) are known to play an important role in photoaging by mediating the degradation of extracellular matrix proteins. To develop a new anti-aging agent for cosmetics from natural products, Melothria heterophylla (Lour.) Cogn. was selected for its antioxidant activity and inhibitory effect on expression of MMP-1 in UVA-irradiated human skin fibroblasts. Two compounds (compounds 1 and 2 ) were isolated from an ethyl acetate soluble fraction of the ethanolic extracts; they were identified as 1,2,4,6-tetra-O-galloyl-beta-(D)-glucopyranose (1) and 3,4,5-trihydroxybenzoic acid (2). These compounds were found to scavenge radicals and reactive oxygen species (ROS) and were measured to have SC50 values of 3.9 microM and 13.3 microM against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and values of 4.3 microM and 4.0 microM against superoxide radicals in the xanthine/xanthine oxidase system, respectively. Compounds 1 and 2 showed a dose-dependent inhibitory effect on the expression and activity of MMP-1 in UVA-induced human skin fibroblasts, but no inhibition of MMP-1 mRNA expression. Therefore, we concluded that compounds 1 and 2 significantly inhibited MMP-1 expression at the protein level. Also, these compounds were determined to have a potent antioxidant activity. From these results, we suggest that these compounds might be useful as a new anti-aging agent for photodamaged skin, but the in vitro findings must be verified in in vivo studies.

  14. Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts

    SciTech Connect

    Roza, L.; Vermeulen, W.; Bergen Henegouwen, J.B.; Eker, A.P.; Jaspers, N.G.; Lohman, P.H.; Hoeijmakers, J.H. )

    1990-03-15

    UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

  15. The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts.

    PubMed

    Schultz, Francisca; Hasan, Alveera; Alvarez-Laviada, Anita; Miragoli, Michele; Bhogal, Navneet; Wells, Sarah; Poulet, Claire; Chambers, Jenny; Williamson, Catherine; Gorelik, Julia

    2016-01-01

    Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts. Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential. We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells. UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts

  16. 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

  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.

  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.

  20. 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.

  1. Effects of gender and body weight on fibroblast growth factor 23 responsiveness to estimated dietary phosphorus.

    PubMed

    Ohta, Hiroyuki; Sakuma, Masae; Suzuki, Akitsu; Morimoto, Yuuka; Ishikawa, Makoto; Umeda, Minako; Arai, Hidekazu

    2016-01-01

    Fibroblast growth factor 23 (FGF23) is a molecule involved in regulating phosphorus homeostasis. Although some studies indicated an association between serum FGF23 levels and sex, the association has not been fully investigated. The purpose of this study was to evaluate whether sex could influence FGF23 responsiveness to dietary phosphorus intake in healthy individuals. Thirty two healthy subjects between 21 and 28 years were recruited for this study. Subjects performed 24-hour urine collection and blood samples were collected. We estimated phosphorus intake (UC-P) from the urine collection (UC), and evaluated any association between UC-P and serum FGF23 levels. Subsequently, we compared serum FGF23 levels between males and females. Positive correlation was observed between UC-P and serum FGF23 levels. Serum FGF23 levels were significantly higher in males than in females. Serum FGF23 levels/UC-P was significantly higher in females than in males. There was no significant difference in serum FGF23 levels/UC-P/BW between the male and female groups. Our results indicate that there was no gender difference between FGF23 responsiveness to phosphorus intake per body weight.

  2. 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.

  3. The effects of Ce3+ and Ce4+ on the stability of fibroblast growth factor-2

    NASA Astrophysics Data System (ADS)

    Sun, Liwei; Feng, Hao; Jiang, Rui; Niu, Liping; Song, Yu; Feng, Kai; Qi, Chao

    2010-11-01

    The interaction between tri or tetravalent cerium ions and basic fibroblast growth factor (FGF-2) at 0.1-6: 1 molar ratio under physiological condition was studied by fluorescence and CD spectrum. The different spectra alterations of FGF-2 induced by Ce3+ and Ce4+ showed that Ce3+ and Ce4+ caused different conformational changes of FGF-2 respectively, though both of them destabilized the protein. The instability of FGF-2 in the presence of Ce3+ is involved in the oxidation of its free cystein of protein, but that this treatment nearly does not affect the biological activity. As to Ce4+, it not only induced the conformational changes of protein but also inhibits its activity in a dose-dependent manner, which could be relative to the electrostatic repulsion between Ce4+ and its basic amino acid residues (pI=9.6) or the specific binding of Ce4+ to deprotonated amino acid residues. The interesting results would be helpful to investigate the problem of the stability of proteins.

  4. 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

  5. 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.

  6. [Manifestation of the adaptive response and bystander-effect of C3H10T1/2 fibroblasts irradiated by protons and gamma-rays].

    PubMed

    Voskanian, K Sh; Mitsyn, G V; Gaevskiĭ, V N

    2009-01-01

    Adaptive response and bystander-effect were studied in mice fibroblasts irradiated by gamma-rays and protons with the energy of 150 MeV Monolayer of fibroblasts cultivated on the wall of a plastic vial first were exposed to 2 and 4 cGy of ionizing radiation (presumably adaptive doses) and later, after 40-min. or 16-hr. period at 37 degrees C, to damaging 4 Gy. To study the bystander-effect, either the whole vial surface (25 cm2) or central area (1 cm2) were irradiated by a beam of protons. The results showed that the preliminary gamma-irradiation 40-min. or 16-hr. before exposure to the damaging dose equally alleviates the harmful effect of protons on fibroblasts. The adaptive response was observed as in the cells subjected to the direct irradiation by protons at 4 Gy, so in bystander-cells. When protons were used for adaptive irradiation, the response was visible only to the dose of 4 cGy in fibroblasts exposed to gamma-radiation 16 hrs. later. In all the rest cases, proton- and gamma-induced damages added together. Besides, the experiments showed that the adaptive effect of protons is passed on to bystander-cells. Adaptive and damaging gamma-irradiation evoked the response invariably.

  7. Involvement of B2 receptor in bradykinin-induced proliferation and proinflammatory effects in human nasal mucosa-derived fibroblasts isolated from chronic rhinosinusitis patients.

    PubMed

    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.

  8. Dental restorative biomaterials induce glutathione depletion in cultured human gingival fibroblast: protective effect of N-acetyl cysteine.

    PubMed

    Stanislawski, L; Soheili-Majd, E; Perianin, A; Goldberg, M

    2000-09-05

    Eight biomaterials eluted from four different types of dental restorative biomaterials, that is, from glass-ionomer cement (GIC: Ketac-fil and Fuji II), resin-modified glass ionomer cement (RM-GIC: Fuji II LC and Photac-fil), composite (Z100 MP and Tetric-flow), and compomer (Compoglass F and F-2000), were studied for their cytotoxic properties in relation to glutathione (GSH) content in cultured human gingival fibroblasts. Z100 MP, Tetric-flow, and Compoglass F were less cytotoxic than the others, with a toxic concentration of 50% (TC 50) > 24% (of eluate), as determined by the MTT test. F-2000, Tetric-flow, and the other biomaterials were relatively more cytotoxic (TC 50 = 9-16%). With the exception of Z100 MP, all the biomaterials induced a depletion of cellular glutathione (GSH) that was variable depending upon the biomaterial eluates. The strongest GSH depletion was with F-2000, Fuji II, and Photac-fil. GSH depletion, with Compoglass and F-2000, was rapid-detectable after one h of cell treatment and complete within 3 h-whereas a longer period of incubation was required for the other biomaterials. Interestingly, the drug cytotoxic effects induced by all the biomaterials were prevented by cell treatment with the antioxidant N-acetylcysteine (NAC). This study provides evidence that the cytotoxic property of dental restorative biomaterials is associated with depletion of the glutathione level in gingival fibroblasts. While the molecular mechanisms of this phenomenon require further investigations, our data suggest that NAC may be useful in preventing the cellular damage induced by dental restorative biomaterials.

  9. Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression

    PubMed Central

    Liu, Cheng-Hai; Hu, Yi-Yang; Wang, Xiao-Ling; Xu, Lie-Ming; Liu, Ping

    2000-01-01

    AIM: To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro. METHODS: NIH/3T3 fibroblasts were cultured routinely, and incubated with 10-4 mol/L-10-7 mol/L SA-A for 22 h. The cell viability was assayed by [3H]proline incorporation, cell proliferation by [3H]TdR incorporation, cell collagen synthetic rate was measured with [3H]proline impulse and collagenase digestion method. The total RNA was prepared from the control cells and the drug treated cells respectively, and α (1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR. RESULTS: 10-4 mol/L SA-A decreased cell viability and exerted some cytotoxiciy, while 10-5 mol/L-10-7 mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10-6 mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10-5 mol/L-10-6 mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10-5 mol/L and 10-6 mol/L SA-A could decrease α (1) I pro-collagen mRNA expression remarkably. CONCLUSION: SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I pro-collagen gene expression, which may be one of the main mechanisms of the drug. PMID:11819598

  10. Essential Oils from Ugandan Medicinal Plants: In Vitro Cytotoxicity and Effects on IL-1β-Induced Proinflammatory Mediators by Human Gingival Fibroblasts

    PubMed Central

    Bwanga, Freddie; Joloba, Moses; Borg-Karlson, Anna-Karin; Yucel-Lindberg, Tülay; Obua, Celestino

    2016-01-01

    The study investigated cytotoxicity of essential oils from four medicinal plants (Bidens pilosa, Ocimum gratissimum, Cymbopogon nardus, and Zanthoxylum chalybeum) on human gingival fibroblasts and their effects on proinflammatory mediators' secretion. Cytotoxicity of essential oils was investigated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Effects of essential oils at subcytotoxicity concentrations on interleukin- (IL-) 6, IL-8, and prostaglandin E2 (PGE2) secretions by gingival fibroblasts treated with IL-1β (300 pg/mL) were evaluated by ELISA and EIA. IC50 values of the essential oils ranged from 26 μg/mL to 50 μg/mL. Baseline and IL-1β-induced secretion of PGE2 was inhibited by treatment with essential oil from O. gratissimum. Essential oils from B. pilosa and C. nardus had synergistic effects with IL-1β on PGE2 seceretion. In conclusion, the study suggests that essential oil from O. gratissimum decreases gingival fibroblasts secretion of PGE2, while essential oils from B. pilosa and C. nardus increase PGE2 secretion. Essential oil from Z. chalybeum was the most cytotoxic, while oil from C. nardus was the least cytotoxic. Although the clinical significance of these findings remains to be determined, it may be suggested that essential oil from O. gratissimum, applied at subcytotoxicity concentrations, could reduce the participation of gingival fibroblasts in the gingival inflammation and tissue destruction associated with periodontitis. PMID:27807462

  11. 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.

  12. Essential Oils from Ugandan Medicinal Plants: In Vitro Cytotoxicity and Effects on IL-1β-Induced Proinflammatory Mediators by Human Gingival Fibroblasts.

    PubMed

    Ocheng, Francis; Bwanga, Freddie; Almer Boström, Elisabeth; Joloba, Moses; Borg-Karlson, Anna-Karin; Yucel-Lindberg, Tülay; Obua, Celestino; Gustafsson, Anders

    2016-01-01

    The study investigated cytotoxicity of essential oils from four medicinal plants (Bidens pilosa, Ocimum gratissimum, Cymbopogon nardus, and Zanthoxylum chalybeum) on human gingival fibroblasts and their effects on proinflammatory mediators' secretion. Cytotoxicity of essential oils was investigated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Effects of essential oils at subcytotoxicity concentrations on interleukin- (IL-) 6, IL-8, and prostaglandin E2 (PGE2) secretions by gingival fibroblasts treated with IL-1β (300 pg/mL) were evaluated by ELISA and EIA. IC50 values of the essential oils ranged from 26 μg/mL to 50 μg/mL. Baseline and IL-1β-induced secretion of PGE2 was inhibited by treatment with essential oil from O. gratissimum. Essential oils from B. pilosa and C. nardus had synergistic effects with IL-1β on PGE2 seceretion. In conclusion, the study suggests that essential oil from O. gratissimum decreases gingival fibroblasts secretion of PGE2, while essential oils from B. pilosa and C. nardus increase PGE2 secretion. Essential oil from Z. chalybeum was the most cytotoxic, while oil from C. nardus was the least cytotoxic. Although the clinical significance of these findings remains to be determined, it may be suggested that essential oil from O. gratissimum, applied at subcytotoxicity concentrations, could reduce the participation of gingival fibroblasts in the gingival inflammation and tissue destruction associated with periodontitis.

  13. Effect of the combination of basic fibroblast growth factor and cysteine on corneal epithelial healing after photorefractive keratectomy in patients affected by myopia

    PubMed Central

    Meduri, Alessandro; Scorolli, Lucia; Scalinci, Sergio Zaccaria; Grenga, Pier Luigi; Lupo, Stefano; Rechichi, Miguel; Meduri, Enrico

    2014-01-01

    Background: This study sought to evaluate the effect of basic fibroblast growth factor eye drops and cysteine oral supplements on corneal healing in patients treated with photorefractive keratectomy (PRK). Materials and Methods: One hundred and twenty patients treated bilaterally with PRK for myopia were enrolled at one of two eye centers (Clinica Santa Lucia, Bologna, Italy and Department of Ophthalmology, University of Magna Graecia, Catanzaro, Italy) and were treated at the former center. Sixty patients included in the study group (Group 1) were treated postoperatively with topical basic fibroblast growth factor plus oral L-cysteine supplements, whereas 60 subjects included in the control group (Group 2) received basic fibroblast growth factor eye drops. We recorded the rate of corneal re-epithelialization and patients were followed-up every 30 days for 6 months. Statistical analyses were performed on the collected data. Results: The eyes in Group 1 demonstrated complete re-epithelialization at Day 5, whereas the eyes in Group 2 achieved this status on Day 6. No side-effects were reported. Conclusions: Patients treated with basic fibroblast growth factor eye drops and L-cysteine oral supplements benefit from more rapid corneal re-epithelialization. In human eyes, this combination treatment appeared to be safe and effective in accelerating corneal surfacing after surgery. Financial Disclosure: No author has any financial or proprietary interest in any material or method used in this study. Trial Registration: Current Controlled Trials ISRCTN73824458. PMID:24145571

  14. 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

    2016-09-02

    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.

  15. Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae.

    PubMed

    Noubhani, A; Bunoust, O; Rigoulet, M; Thevelein, J M

    2000-07-01

    In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Delta spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Delta strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein-protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also

  16. Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schlenker, Heiter; Brenner, Rolf E; Fiedler, Jörg; Friemert, Benedikt; Dürselen, Lutz; Ignatius, Anita

    2012-10-01

    The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.

  17. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    PubMed

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  18. 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.

  19. Dose-dependent effects of allopurinol on human foreskin fibroblast cells and human umbilical vein endothelial cells under hypoxia.

    PubMed

    Sun, Yu; George, Jacob; Rocha, Sonia

    2015-01-01

    Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells.

  20. Surfactant protein A is a principal and oxidation-sensitive microbial permeabilizing factor in the alveolar lining fluid.

    PubMed

    Kuzmenko, Alexander I; Wu, Huixing; Wan, Sijue; McCormack, Francis X

    2005-07-08

    We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A+/+ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A-/- animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A+/+ mice increased membrane permeability more than those from SP-A-/- mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A+/+ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.

  1. Effects of basic fibroblast growth factor and insulin-like growth factor on cultured cartilage cells from skate Raja porasa

    NASA Astrophysics Data System (ADS)

    Fan, Tingjun; Jin, Lingyun; Wang, Xiaofeng

    2003-12-01

    Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) on cartilage cells from proboscis of skate, Raja porasa Günther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24°C. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF-II at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-II was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF-II together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-II together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7.

  2. Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells.

    PubMed

    Hatziapostolou, Maria; Polytarchou, Christos; Katsoris, Panagiotis; Courty, Jose; Papadimitriou, Evangelia

    2006-10-27

    Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.

  3. 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

  4. Cytotoxic effect of silorane and methacrylate based composites on the human dental pulp stem cells and fibroblasts

    PubMed Central

    Shafiei, Fereshteh; Razmkhah, Mahboobeh; Attar, Armin; Alavi, Ali A.

    2014-01-01

    Objectives: The aim of this study was to compare the cytotoxic effect of a methacrylate-based and a silorane-based composite on the human dental pulp stem cells (DPSCs) versus human dental pulp fibroblasts (DPFs). Study Design: Samples of the Filtek Z250 and P90 were polymerized and immersed in the culture medium to obtain extracts after incubation for one, seven and 14 days. Magnetic cell sorting based on the CD146 expression was performed to purify DPSCs and DPFs. After incubation of both cells with the extracts, cytotoxicity was determined using the MTT test. Results: For the extracts of first and seventh day, both composites showed significantly lower cytotoxicity on DPSCs than DPFs (p=0.003). In addition, there was a significant difference in the time-group interaction of both materials indicating different cytotoxic behaviours (p=0.014). In contrast to Z250, exposure to the 14th day extract of P90 resulted in higher cell viability compared to that of day seven. Conclusions: DPSCs are less susceptible to the cytotoxic effect of the composites than DPFs. Compared to Z250, the cytotoxic effect of silorane-based composite decreases as the time passes on. This difference should be considered, particularly in deep cavities, in order to preserve the regenerative capacity of the pulp. Key words:Composite resins, Dental pulp, Mesenchymal Stromal Cells, Silorane, Toxicology. PMID:24608214

  5. Mobile phone signal exposure triggers a hormesis-like effect in Atm(+/+) and Atm(-/-) mouse embryonic fibroblasts.

    PubMed

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-11-18

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm(+/+)) or deficient (Atm(-/-)) ATM. In Atm(+/+) MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm(-/-) MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF.

  6. Effects of some new antidepressant drugs on the glucocorticoid receptor-mediated gene transcription in fibroblast cells.

    PubMed

    Augustyn, Matylda; Otczyk, Magdalena; Budziszewska, Bogusława; Jagła, Grzegorz; Nowak, Wojciech; Basta-Kaim, Agnieszka; Jaworska-Feil, Lucylla; Kubera, Marta; Tetich, Magdalena; Leśkiewicz, Monika; Lasoń, Władysław

    2005-01-01

    Antidepressant drugs are thought to counteract effects of hypercortisolemia, frequently associated with depression, by lowering cortisol level and by modifying the function of glucocorticoid receptors (GR). Indeed, classical antidepressants inhibit corticosteroid-induced gene transcription in cell cultures. The aim of the present study was to investigate effects of new generation antidepressant drugs on GR function in mouse fibroblast cells (L929), stably transfected with mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) plasmid (LMCAT cells). It has been found that reboxetine (at 10 and 30 microM), venlafaxine, citalopram and mirtazapine (at 30 microM), but not milnacipran, in statistically significant manner inhibited corticosterone-induced gene transcription. However, the effects of new generation antidepressant drugs were weaker than those evoked by imipramine, which was active already at 3 microM concentration. Further studies on the mechanism of antidepressant action on GR function revealed that protein kinase C, but not mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK-3) and protein kinase B (PKB, Akt) play a role in this phenomenon.

  7. Mobile phone signal exposure triggers a hormesis-like effect in Atm+/+ and Atm−/− mouse embryonic fibroblasts

    PubMed Central

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-01-01

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm+/+) or deficient (Atm−/−) ATM. In Atm+/+ MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm−/− MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF. PMID:27857169

  8. Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier

    PubMed Central

    Bonakdar, Mohammad; Wasson, Elisa M.; Lee, Yong W.; Davalos, Rafael V.

    2016-01-01

    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

  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.

  10. Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.

    PubMed

    Daniels, Robert; Rusan, Nasser M; Wilbuer, Anne-Kathrin; Norkin, Leonard C; Wadsworth, Patricia; Hebert, Daniel N

    2006-07-01

    Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

  11. 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.

  12. Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons.

    PubMed

    Clerc, Pascaline; Polster, Brian M

    2012-01-01

    Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.

  13. 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.

  14. Effects of 915 nm GaAs diode laser on mitochondria of human dermal fibroblasts: analysis with confocal microscopy.

    PubMed

    Belletti, Silvana; Uggeri, Jacopo; Mergoni, Giovanni; Vescovi, Paolo; Merigo, Elisabetta; Fornaini, Carlo; Nammour, Samir; Manfredi, Maddalena; Gatti, Rita

    2015-01-01

    Low-level laser therapy (LLLT) is widely used in tissue regeneration and pain therapy. Mitochondria are supposed to be one of the main cellular targets, due to the presence of cytochrome C oxidase as photo-acceptor. Laser stimulation could influence mitochondria metabolism affecting mainly transmembrane mitochondrial potential (Δψm). The aim of our study is to evaluate "in vitro" the early mitochondrial response after irradiation with a 915 GaAs laser. Since some evidences suggest that cellular response to LLLT can be differently modulated by the mode of irradiation, we would like to evaluate whether there are changes in the mitochondrial potential linked to the use of the laser treatments applied with continuous wave (CW) in respect to those applied with pulsed wave (PW). In this study, we analyzed effects of irradiation with a 915-nm GaAs diode laser on human dermal fibroblast. We compared effects of irradiation applied with either CW or PW at different fluences 45-15-5 J/cm(2) on Δψm. Laser scanning microscopy (LSM) was used in living cells to detect ROS (reactive oxygen species) using calcein AM and real-time changes of and Δψm following distribution of the potentiometric probe tetramethylrhodamine methyl ester (TMRM). At higher doses (45-15 J/cm(2)), fibroblasts showed a dose-dependent decrement of Δψm in either the modalities employed, with higher amplitudes in CW-treated cells. This behavior is transient and not followed by any sign of toxicity, even if reactive oxygen species generation was observed. At 5 J/cm(2), CW irradiation determined a little decrease (5%) of the baseline level of Δψm, while opposite behavior was shown when cells were irradiated with PW, with a 10% increment. Our results suggest that different responses observed at cellular level with low doses of irradiation, could be at the basis of efficacy of LLLT in clinical application, performed with PW rather than CW modalities.

  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.

  16. Demonstration of inhibitory effect of oral shark cartilage on basic fibroblast growth factor-induced angiogenesis in the rabbit cornea.

    PubMed

    González, R P; Soares, F S; Farias, R F; Pessoa, C; Leyva, A; de Barros Viana, G S; Moraes, M O

    2001-02-01

    Several angiogenic inhibitors have been obtained from shark cartilage, some of these are currently in clinical trials for assessment of safety and therapeutic efficacy in humans. Still, shark cartilage taken orally is commonly used in alternative and complimentary medicine for various ailments including serious diseases such as cancer. However, only few studies of oral shark cartilage have demonstrated pharmacological effects in experimental animals or patients, to indicate safe doses with sufficient bioavailability. In the present study we demonstrated the antiangiogenic properties of oral shark cartilage in the rabbit cornea model. Slow-release, polymethylmetacrylate pellets containing basic fibroblast growth factor (bFGF) were surgically implanted in the rabbit cornea to stimulate neovascularization scored by stereo microscopy. Powdered shark cartilage (PSC; commercial product) was tested orally along with a water-soluble fraction (WSF) of this cartilage product which was tested by local application. Animals were treated with oral dosages of 100 mg/kg PSC or 200 mg/kg thalidomide as positive control. Pellets containing WSF (50, 100 or 200 microg/pellet) or bFGF-inhibitor pentosan polysulfate were implanted adjacent to the bFGF pellet. Oral shark cartilage inhibited bFGF-induced angiogenesis, as did oral thalidomide, in this in vivo model. WSF and pentosan polysulfate was shown to block neovascularization in the cornea when applied locally. This study demonstrates that in the rabbit, oral shark cartilage appears to produce systemic levels of angiogenesis inhibitors that can exert their effect at the cornea.

  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. Modulating fibroblast cell proliferation with functionalized poly(methyl methacrylate) based copolymers: chemical composition and monomer distribution effect.

    PubMed

    El Khadali, Fatima; Hélary, Gérard; Pavon-Djavid, Graciela; Migonney, Véronique

    2002-01-01

    Poly(methyl methacrylate)-based terpolymers bearing sulfonate and carboxylate groups have been synthesized by radical copolymerization leading to polymers with random distributions of ionic monomer units. Fibroblast cells were seeded on terpolymers of various molar compositions of ionic groups. Kinetics of the cell proliferation were examined and systematically compared to the nonfunctionalized control polymer, poly(methyl methacrylate). Modulation of cell proliferation was observed on 15% ionic monomer content copolymers of various compositions (R = COO(-)/(COO(-) + SO(3)(-)) and varies from 0 to 1). The inhibition percentage of cell proliferation calculated for each polymer by comparison to the cell proliferation on the control was plotted against R and gave a maximum value for R close to 0.55. Copolymers with ionic group contents higher or lower than 15% exhibit inhibition percentages fitting with those previously observed for the same R values, showing that the hydrophilic properties are not sufficient to explain the modulation effect of this material toward cells. Moreover, for each polymer tested, cells, even if inhibited in growth, were shown to be viable, indicating that the synthesized terpolymers exhibit cytostatic properties excluding any cytotoxic effect. Such polymers may be used for the fabrication of biocompatible intraocular lenses and prevent secondary cataract.

  19. A Pilot Study of the Photoprotective Effects of Strawberry-Based Cosmetic Formulations on Human Dermal Fibroblasts.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Yuliett; Afrin, Sadia; Alvarez-Suarez, José Miguel; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Bompadre, Stefano; Quiles, José Luis; Mezzetti, Bruno; Giampieri, Francesca

    2015-08-04

    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.

  20. Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films--effects on fibroblast spreading.

    PubMed

    Satriano, Cristina; Messina, Grazia M L; Marino, Clara; Aiello, Ivana; Conte, Enrico; La Mendola, Diego; Distefano, Donatella A; D'Alessandro, Franca; Pappalardo, Giuseppe; Impellizzeri, Giuseppe

    2010-01-15

    The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV-ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.

  1. 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

  2. Toxic effects of some conifer resin acids and tea tree oil on human epithelial and fibroblast cells.

    PubMed

    Söderberg, T A; Johansson, A; Gref, R

    1996-02-22

    The present study was undertaken to assess and compare the in vitro cytotoxic effects of three resin acid analogues: dehydrobietic acid, podocarpic acid, O-methylpodocarpic acid; an essential oil from Australia (tea tree oil); and tapped oleoresin from Thailand, on human epithelial and fibroblast cells, using a quantitative neutral red spectrophotometric assay. All of the investigated compounds except for tea tree oil exhibited a cytotoxic activity which was proportional to their concentrations and time of exposure up to 24 h, i.e. higher concentrations and longer time of exposure caused increased cell death. Dehydroabietic acid and the oleoresin were the most toxic compounds followed by O-methylpodocarpic acid, whereas podocarpic acid and tea tree oil showed a lower level of toxicity. On the basis on these findings it is concluded that an isopropyl group on the aromatic C-ring is of great importance for the cytotoxicity of the tested abietane resin acids, thus indicating that the cytotoxic activity of oleoresins most probably is caused by synergistic or additive effects of resin acids. The results from this work support the view that antibacterial activity parallels cytotoxic activity which suggests a similar mode of action, most probably exerted by membrane-associated reactions.

  3. 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

  4. 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.

  5. 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.

  6. Cardiac Fibrosis: The Fibroblast Awakens

    PubMed Central

    Travers, Joshua G.; Kamal, Fadia A.; Robbins, Jeffrey; Yutzey, Katherine E.; Blaxall, Burns C.

    2016-01-01

    Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of this cell population impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis. PMID:26987915

  7. (Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture.

    PubMed

    van Meeuwen, J A; Korthagen, N; de Jong, P C; Piersma, A H; van den Berg, M

    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(50)s decreased in the following order: estadiol (4*10(-3) nM)>biochanin A (9 nM)>genistein (32 nM)>testosterone (46 nM)>naringenin (287 nM)>apigenin (440 nM)>chrysin (4 microM). The potency to inhibit aromatase derived from their IC(50)s decreased in the following order: chrysin (1.5 microM)>naringenin (2.2 microM)>genistein (3.6 microM)>apigenin (4.1 microM)>biochanin A (25 microM)>quercetin (30 microM). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1-10 microM). 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 of individuals using food

  8. (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

  9. Cristae remodeling causes acidification detected by integrated graphene sensor during mitochondrial outer membrane permeabilization

    PubMed Central

    Pham, Ted D.; Pham, Phi Q.; Li, Jinfeng; Letai, Anthony G.; Wallace, Douglas C.; Burke, Peter J.

    2016-01-01

    The intrinsic apoptotic pathway and the resultant mitochondrial outer membrane permeabilization (MOMP) via BAK and BAX oligomerization, cytochrome c (cytc) release, and caspase activation are well studied, but their effect on cytosolic pH is poorly understood. Using isolated mitochondria, we show that MOMP results in acidification of the surrounding medium. BAK conformational changes associated with MOMP activate the OMA1 protease to cleave OPA1 resulting in remodeling of the cristae and release of the highly concentrated protons within the cristae invaginations. This was revealed by utilizing a nanomaterial graphene as an optically clear and ultrasensitive pH sensor that can measure ionic changes induced by tethered mitochondria. With this platform, we have found that activation of mitochondrial apoptosis is accompanied by a gradual drop in extra-mitochondrial pH and a decline in membrane potential, both of which can be rescued by adding exogenous cytc. These findings have importance for potential pharmacological manipulation of apoptosis, in the treatment of cancer. PMID:27786282

  10. Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis.

    PubMed

    Yu, L; O'Sullivan, D J

    2014-02-01

    Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

  11. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

    PubMed

    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  12. Improvement on D-xylose to Xylitol Biotransformation by Candida guilliermondii Using Cells Permeabilized with Triton X-100 and Selected Process Conditions.

    PubMed

    Cortez, Daniela Vieira; Mussatto, Solange I; Roberto, Inês Conceição

    2016-11-01

    Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl2·6H2O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

  13. Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation

    PubMed Central

    1989-01-01

    Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation. PMID:2507555

  14. 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

  15. A minority of carcinoma cells producing acidic fibroblast growth factor induces a community effect for tumor progression.

    PubMed Central

    Jouanneau, J; Moens, G; Bourgeois, Y; Poupon, M F; Thiery, J P

    1994-01-01

    It is generally accepted that primary tumors become heterogeneous as a consequence of tumor-cell genetic instability. Clonal dominance has been shown to occur in some experimental models allowing a subpopulation of cells to overgrow the primary heterogeneous tumor and to metastasize. Alternatively, interactions among coexisting tumor subpopulations may contribute to the emergence of a malignant invasive primary solid tumor. We asked the question whether emergence of carcinoma cells producing a growth/dissociating factor within a tumor cell population may be a determinant for tumor progression and for clonal dominance. To mimic such a situation, we have investigated the impact of tumor subpopulation heterogeneity in an in vivo model in which mixtures of carcinoma cells that differ in their ability to produce acidic fibroblast growth factor are injected into nude mice. Our data indicate that a growth-factor-producing cell subpopulation can confer increased tumorigenicity to an entire cell population and subsequently elicit a shorter delay for appearance of metastasis. A community effect via cell interactions may account for a heterogeneous tumor cell population rather than clonal dominance during progression of certain tumor types. Images Fig. 3 PMID:7506417

  16. Effects of fiber orientation and diameter on the behavior of human dermal fibroblasts on electrospun PMMA scaffolds.

    PubMed

    Liu, Ying; Ji, Yuan; Ghosh, Kaustabh; Clark, Richard A F; Huang, Lei; Rafailovich, Miriam H

    2009-09-15

    We used the electrospinning technique to produce fibrous scaffolds of poly(methyl methacrylate) (PMMA). Using a rotating drum, we aligned the fibers and formed multilayered structures where both the fiber spacing and pore size could be varied. We then plated adult human dermal fibroblasts and studied the effect of fiber diameter and orientation on the cell conformation, integrin receptor expression, proliferation, and migration. We found that a critical diameter minimum diameter existed, D0 = 0.97 microm for cell orientation to occur. For D < D0, no big difference in aspect ratio was observed relative to the control samples on PMMA thin film. Hence, we could fabricate substrate patterned with fibers of different diameters where different cell conformations coexisted on the same scaffold. On the other hand, staining for vinculin proteins in the cells indicated that on large diameter fibers and on flat surfaces, the integrin receptors followed the cell perimeter. On the very small diameter surfaces, the receptors were distributed uniformly along the cell. Cell dynamics studies indicated that the proliferation and migration were also affected by the fiber orientation.

  17. Phenolic composition and antioxidant effect of aqueous extract of Arisaema cum Bile, the Oriental Herb Medicine, in human fibroblast cells.

    PubMed

    Ahn, Chang-Bum; Shin, Tai-Sun; Seo, Hye Kyoung; Je, Jae-Young

    2012-08-01

    Phenolic composition and antioxidant activities of the aqueous extract of Arisaema cum Bile, which is widely used as a folk medicine in Korea, were determined. Phenolic composition profile revealed that the aqueous extract is rich in sinapic acid (13.14 mg/100 g extract), catechin (9.88 mg/100 g extract), neohesperidin (7.38 mg/100 g extract), and chlorogenic acid (3.64 mg/100 g extract). The aqueous extract effectively scavenged toward 2,2-diphenyl-1-picrylhydrazyl (90.63%), hydrogen peroxide (98.13%), and hydroxyl radical (59.62%) at 2.0 mg/mL, and also showed high reducing power. In cytotoxic evaluation, the aqueous extract exhibited no significant cytotoxicity in human fibroblast, and it also exhibited appreciable suppression of intracellular reactive oxygen species and inhibition of lipid peroxidation. In addition, the aqueous extract upregulated the level of glutathione in a dose-dependent manner. Taken together, the aqueous extract of Arisaema cum Bile could be considered as a potential natural source that may be useful for curing diseases arising from oxidative deterioration.

  18. Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

    PubMed Central

    Sproul, Christopher D.; Mitchell, David L.; Rao, Shangbang; Ibrahim, Joseph G.; Kaufmann, William K.; Cordeiro-Stone, Marila

    2015-01-01

    This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine–pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA–UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR. PMID:24148148

  19. 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.

  20. Role of human pulp fibroblasts in angiogenesis.

    PubMed

    Tran-Hung, L; Mathieu, S; About, I

    2006-09-01

    After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

  1. Induction of bystander effects by UVA, UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species.

    PubMed

    Widel, Maria; Krzywon, Aleksandra; Gajda, Karolina; Skonieczna, Magdalena; Rzeszowska-Wolny, Joanna

    2014-03-01

    Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role

  2. Inhibitory Effect of Progesterone on Cervical Tissue Formation in a Three-Dimensional Culture System with Human Cervical Fibroblasts1

    PubMed Central

    House, Michael; Tadesse-Telila, Serkalem; Norwitz, Errol R.; Socrate, Simona; Kaplan, David L.

    2013-01-01

    ABSTRACT Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10−8 M), progesterone (10−7 or 10−6 M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10−8 M) and five conditions: vehicle; 10−9, 10−8, or 10−7 M progesterone; or 10−7 M progesterone plus 10−6 M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix. PMID:24285720

  3. Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts

    SciTech Connect

    Snyder, R.D.; Matheson, D.W.

    1985-01-01

    An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

  4. 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

  5. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

    PubMed Central

    Tanaka, Miyuki; Misawa, Eriko; Yamauchi, Koji; Abe, Fumiaki; Ishizaki, Chiaki

    2015-01-01

    Background Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods First, we investigated the capability of Aloe sterols (cycloartenol and lophenol) to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP) containing 40 μg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake period. Conclusion The present study confirms that daily oral Aloe sterol-containing AVGP significantly reduced facial wrinkles in women aged ≥40 years, and Aloe sterols stimulate collagen and hyaluronic acid production by human dermal fibroblasts. PMID:25759593

  6. Effects of JWH015 in cytokine secretion in primary human keratinocytes and fibroblasts and its suitability for topical/transdermal delivery

    PubMed Central

    Bort, Alicia; Alvarado-Vazquez, Perla A; Moracho-Vilrriales, Carolina; Virga, Kristopher G; Gumina, Giuseppe; Asbill, Scott

    2017-01-01

    Background JWH015 is a cannabinoid (CB) receptor type 2 agonist that produces immunomodulatory effects. Since skin cells play a key role in inflammatory conditions and tissue repair, we investigated the ability of JWH015 to promote an anti-inflammatory and pro-wound healing phenotype in human primary skin cells. Methods Human primary keratinocytes and fibroblasts were stimulated with lipopolysaccharide. The mRNA expression of cannabinoid receptors was determined using RT-PCR. The effects of JWH015 (0.05, 0.1, 0.5, and 1 µM) in pro- and anti-inflammatory factors were tested in lipopolysaccharide-stimulated cells. A scratch assay, using a co-culture of keratinocytes and fibroblasts, was used to test the effects of JWH015 in wound healing. In addition, the topical and transdermal penetration of JWH015 was studied in Franz diffusion cells using porcine skin and LC-MS. Results The expression of CB1 and CB2 receptors (mRNA) and the production of pro- and anti-inflammatory factors enhanced in keratinocytes and fibroblasts following lipopolysaccharide stimulation. JWH015 reduced the concentration of major pro-inflammatory factors (IL-6 and MCP-1) and increased the concentration of a major anti-inflammatory factor (TGF-β) in lipopolysaccharide-stimulated cells. JWH015 induced a faster scratch gap closure. These JWH015’seffects were mainly modulated through both CB1 and CB2 receptors. Topically administered JWH015 was mostly retained in the skin and displayed a sustained and low level of transdermal permeation. Conclusions Our findings suggest that targeting keratinocytes and fibroblasts with cannabinoid drugs could represent a therapeutic strategy to resolve peripheral inflammation and promote tissue repair. PMID:28326930

  7. Protective effects of basic fibroblast growth factor in the development of emphysema induced by interferon-γ.

    PubMed

    Lee, Byung-Jae; Moon, Hyung-Geun; Shin, Tae-Seop; Jeon, Seong Gyu; Lee, Eun-Young; Gho, Yong Song; Lee, Chun Geun; Zhu, Zhou; Elias, Jack A; Kim, Yoon-Keun

    2011-04-30

    Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-γ in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-β, is important in wound healing. We investigated the role of FGF2 in IFN-γ-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-γ-induced emphysema, lung targeted IFN-γ transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 μg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-γ in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-γ but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-γ-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.

  8. 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.

  9. Effect of ebosin on modulating interleukin-1β-induced inflammatory responses in rat fibroblast-like synoviocytes

    PubMed Central

    Zhang, Yang; Wang, Lifei; Bai, Liping; Jiang, Rong; Guo, Lianhong; Wu, Jianbo; Cheng, Guifang; Zhang, Ren; Li, Yuan

    2016-01-01

    The interleukin-1β-mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways are involved in the pathogenesis of rheumatoid arthritis. Ebosin, a novel exopolysaccharide (EPS), exhibits anti-inflammatory activity in rat collagen-induced arthritis by suppressing the production of tumor necrosis factor-α, interleukin-6 and interleukin-1β. The aim of the present study was to assess the effects of ebosin on NF-κB and MAPK signaling pathways mediated through interleukin-1β in rat fibroblast-like synoviocytes (FLSs). Western blotting showed decreased production of phosphorylated p38, JNK1, JNK2, IKKα, IKKβ and IκB in the cytoplasm and NF-κB in the nucleus upon ebosin treatment. The DNA-binding activity of NF-κB in the cell nucleus was also inhibited by ebosin treatment, as demonstrated using an electrophoresis mobility gel shift assay. Analysis of the results of the immunofluorescence assay also showed a reduced amount of NF-κB in the nucleus of cells affected by ebosin. These results provided evidence for the effects of ebosin on both interleukin-1β-mediated MAPK and NF-κB signaling pathways in rat FLSs. In addition, enzyme-linked immunosorbent assay demonstrated that ebosin reduces the levels of matrix metalloproteinases MMP-1 and MMP-3 and the chemokines, interleukin-8 and RANTES. Thus, the results of the present study provide further evidence for understanding the medicinal activity of ebosin at a molecular level, therefore nominating this EPS as a potential therapeutic candidate for the treatment of rheumatic arthritis.

  10. Effects of miR-223 on expression of IL-1β and IL-6 in human gingival fibroblasts.

    PubMed

    Matsui, Sari; Ogata, Yorimasa

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional expression by translational inhibition or mRNA degradation. miRNAs bind to target mRNAs through partial complementarity, and can regulate many genes. In the present study, we investigated the effects of miR-223 on the expression of inflammatory cytokines in human gingival fibroblasts (HGF). To determine the effects of miR-223 on the expressions of interleukin-1β (IL-1β) and IL-6, HGF were stimulated by IL-1β (1 ng/mL) or tumor necrosis factor-α (TNF-α; 10 ng/mL) and transfected with a miR-223 expression plasmid. Levels of mRNA for IL-1β, IL-6, inhibitor of kappa-B kinase α (IKKα) and mitogen-activated protein kinase phosphatase-5 (MKP-5) were measured by real-time PCR, and levels IL-1β, IL-6 and IKKα protein were determined by enzyme-linked immunosorbent assay and Western blotting. Expression of IL-1β and IL-6 mRNAs was induced by IL-1β and TNF-α and further increased by miR-223 overexpression. IL-1β and TNF-α induced the expression of IL-1β and IL-6 mRNAs, and this was reduced by miR-223 inhibitor. Overexpression of miR-223 decreased the levels of IKKα protein and MKP-5 mRNA in HGF. These findings indicate that miR-223 might control the inflammatory response via IKKα and MKP-5 in periodontal tissue. (J Oral Sci 58, 101-108, 2016).

  11. Effects of passivation treatments on titanium alloy with nanometric scale roughness and induced changes in fibroblast initial adhesion evaluated by a cytodetacher.

    PubMed

    Wang, C-C; Hsu, Y C; Su, F C; Lu, S C; Lee, T M

    2009-02-01

    Passivation treatments of titanium alloy alter not only its nanosurface characteristics of oxides and ion release but also surface roughness (Ra), and wettability as well, where nanosurface characteristics of oxides include chemistries of oxides, amphoteric-OH groups adsorbed on oxides, and oxide thickness. Consequently, the passivation treatment affects the alloy's cyto-comparability. In this study, we polish specimens to achieve nanometric scale roughness. In addition, treatment effects are evaluated for surface topology, roughness, wettability, and responses of fibroblasts consisting of MTT assay, initial adhesion strength, and morphology. The initial adhesion strength is measured using a cyto-detacher that achieves nano-Newton resolution. Results reveal that (1) the treatment effects on the percentage of Ti--OH basic groups and wettability are nearly collinear; (2) the Ra of passivated Ti-6Al-4V ranges from 1.9 to 7.4 nm; (3) the initial adhesion strength of fibroblast ranges from 58 to 143 nN, and it is negatively correlated to the Ra; (4) the passivation results in distinguishable morphologies, which further substantiate the negative correlation between cell initial adhesion force and Ra; and (5) our results fall short of confirming previous reports that found positively charged functional groups promoting fibroblast attachment and spread. Potential causes of the inconsistency are addressed.

  12. Fibroblasts from patients with hereditary cutaneous malignant melanoma are abnormally sensitive to the mutagenic effect of simulated sunlight and 4-nitroquinoline 1-oxide

    SciTech Connect

    Howell, J.N.; Greene, M.H.; Corner, R.C.; Maher, V.M.; McCormick, J.J.

    1984-02-01

    Because of a possible etiologic link between mutations and carcinogenesis, the authors compared fibroblasts derived from skin biopsies of several patients with hereditary cutaneous malignant melanoma and the dysplastic nevus syndrome for sensitivity to the mutagenic and/or cytotoxic effect of broad-spectrum simulated sunlight and of a UV mimetic carcinogen, 4-nitroquinoline 1-oxide (4NQO). The genetic marker was resistant to 6-thioguanine; loss of colony-forming ability was the assay for cytotoxicity. All five strains tested were more sensitive than normal to the killing effect of 4NQO (slopes of survival curves were 2- to 3-fold steeper), but only one strain was hypersensitive to killing by Sun Lamp radiation. Two strains were tested for mutagenicity. The response of each to the mutagenic action of these agents corresponded to its response to cell killing. Both strains were hypermutable after exposure to 4NQO, but only one showed a higher than normal frequency of mutants induced by simulated sunlight. The finding that nonmalignant fibroblasts from patients with a hereditary variant of malignant fibroblasts from patients with a hereditary variant of malignant melanoma are abnormally susceptible to carcinogen-induced mutations suggests that hypersensitivity to mutagens contributes to risk of melanoma in patients. It also supports the somatic cell mutation hypothesis for the origin of cancer. 46 references, 3 figures.

  13. 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

  14. Effects of chitosan-coated fibers as a scaffold for three-dimensional cultures of rabbit fibroblasts for ligament tissue engineering.

    PubMed

    Sarukawa, Junichiro; Takahashi, Masaaki; Abe, Masashi; Suzuki, Daisuke; Tokura, Seiichi; Furuike, Tetsuya; Tamura, Hiroshi

    2011-01-01

    Material selection in tissue-engineering scaffolds is one of the primary factors defining cellular response and matrix formation. In this study, we fabricated chitosan-coated poly(lactic acid) (PLA) fiber scaffolds to test our hypothesis that PLA fibers coated with chitosan highly promoted cell supporting properties compared to those without chitosan. Both PLA fibers (PLA group) and chitosan-coated PLA fibers (PLA-chitosan group) were fabricated for this study. Anterior cruciate ligament (ACL) fibroblasts were isolated from Japanese white rabbits and cultured on scaffolds consisting of each type of fiber. The effects of cell adhesivity, proliferation, and synthesis of the extracellular matrix (ECM) for each fiber were analyzed by cell counting, hydroxyproline assay, scanning electron microscopy and quantitative RT-PCR. Cell adhesivity, proliferation, hydroxyproline content and the expression of type-I collagen mRNA were significantly higher in the PLA-chitosan group than in the PLA group. Scanning electron microscopic observation showed that fibroblasts proliferated with a high level of ECM synthesis around the cells. Chitosan coating improved ACL fibroblast adhesion and proliferation, and had a positive effect on matrix production. Thus, the advantages of chitosan-coated PLA fibers show them to be a suitable biomaterial for ACL tissue-engineering scaffolds.

  15. Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion

    PubMed Central

    Huang, Yong; Lu, Min-Qiang; Li, Hua; Xu, Chi; Yi, Shu-Hong; Chen, Gui-Hua

    2006-01-01

    AIM: To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca2+ entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts. METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca2+ entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca2+]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca2+ entry in adhesion was determined using matrigel-coated culture plates. RESULTS: 8-bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1μmol/L). A similar effect on the Ca2+ entry was observed in 3T3 cells in response to a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also observed, indicating NO/cGMP-regulated Ca2+ entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca2+ concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates. CONCLUSION: NO/cGMP sensitive store-operated Ca2+ entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion. PMID:17006985

  16. Effects of DNA methyltransferase inhibitor RG108 on methylation in buffalo adult fibroblasts and subsequent embryonic development following somatic cell nuclear transfer.

    PubMed

    Sun, H L; Meng, L N; Zhao, X; Jiang, J R; Liu, Q Y; Shi, D S; Lu, F H

    2016-09-02

    Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0.94 ± 0.03 and 0.92 ± 0.05 vs 1.0 ± 0.02, 0.98 ± 0.05, and 0.98 ± 0.09, respectively; P < 0.05). Quantitative polymerase chain reaction revealed that DNMT1 gene expression of fibroblasts administered 10, 20, and 100 mM RG108 was significantly lower than those in the 0 and 5 mM groups (0.2 ± 0.05, 0.18 ± 0.07, and 0.3 ± 0.09 vs 1.0 ± 0.12 and 1.4 ± 0.12, respectively; P < 0.05). Treatment with 20 mM RG108 resulted in the lowest expression levels. Fibroblasts incubated with 20 mM RG108 for 72 h were used as donor cells to generate SCNT embryos. A greater number of such embryos developed into blastocysts compared to the non-treated group (28.9 ± 3.9 vs 15.3 ± 3.4%; P < 0.05). RG108 treatment can modify DNA methylation in buffalo adult fibroblasts and promote development of subsequent SCNT embryos.

  17. Resistin-like molecule-β (RELM-β) targets airways fibroblasts to effect remodelling in asthma: from mouse to man

    PubMed Central

    Sharma, S.; Kierstein, S.; Wu, H. F.; Eid, G.; Haczku, A.; Corrigan, C. J.; Ying, S.

    2016-01-01

    Summary Background RELM-β has been implicated in airways inflammation and remodelling in murine models. Its possible functions in human airways are largely unknown. The aim was to address the hypothesis that RELM-β plays a role in extracellular matrix deposition in asthmatic airways. Methods The effects of RELM-β gene deficiency were studied in a model of allergen exposure in mice sensitised and challenged with Aspergillus fumigatus (Af). RELM-β expression was investigated in bronchial biopsies from asthmatic patients. Direct regulatory effects of RELM-β on human lung fibroblasts were examined using primary cultures and the MRC5 cell line in vitro. Results Sensitisation and challenge of wild-type mice with Af-induced release of RELM-β with a time course coincident with that of procollagen in the airways. Af-induced expression of mRNA encoding some, but not all ECM in the lung parenchyma was attenuated in RELM-β−/− mice. RELM-β expression was significantly increased in the bronchial submucosa of human asthmatics compared with controls, and its expression correlated positively with that of fibronectin and α-smooth muscle actin. In addition to epithelial cells, macrophages, fibroblasts and vascular endothelial cells formed the majority of cells expressing RELM-β in the submucosa. Exposure to RELM-β increased TGF-β1, TGF-β2, collagen I, fibronectin, smooth muscle α-actin, laminin α1, and hyaluronan and proteoglycan link protein 1 (Hapl1) production as well as proliferation by human lung fibroblasts in vitro. These changes were associated with activation of ERK1/2 in MRC5 cells. Conclusion The data are consistent with the hypothesis that elevated RELM-β expression in asthmatic airways contributes to airways remodelling at least partly by increasing fibroblast proliferation and differentiation with resulting deposition of extracellular matrix proteins. PMID:25545115

  18. Chlorpromazine as permeabilizer and reagent for detection of microbial peroxidase and peroxidaselike activities.

    PubMed Central

    Galeazzi, L; Turchetti, G; Grilli, G; Groppa, G; Giunta, S

    1986-01-01

    Chlorpromazine was used to perform a test for the detection of microbial peroxidase activities. The compound acts as both a cell permeabilizer and a reagent in the procedure developed which allows the detection of peroxidase and peroxidase like reactions both semiquantitatively in whole cell determinations and quantitatively in cell-free supernatants. PMID:3539020

  19. 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

  20. A Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate in Permeabilized Cells

    PubMed Central

    Kawamata, Hibiki; Starkov, Anatoly A; Manfredi, Giovanni; Chinopoulos, Christos

    2010-01-01

    We have previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg2+] reported by a Mg2+-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg2+. In this manuscript we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides, such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity and myosin ATPase activity. Here we report that addition of BeF3− and Na3VO4 to media containing digitonin-permeabilized cells inhibit all ATP-ADP utilizing reactions, except the ANT-mediated mitochondrial ATP-ADP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F1Fo-ATPase, due to its sensitivity to BeF3− and Na3VO4. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler, and expressed as a function of citrate synthase activity per total amount of protein. PMID:20691655

  1. Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization

    PubMed Central

    Kaneko, Toshiro; Sasaki, Shota; Takashima, Keisuke; Kanzaki, Makoto

    2017-01-01

    Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O2•−), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of •OHaq, resulting from the center-peaked distribution of •OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization. PMID:28163376

  2. Analysis of Soluble Protein Entry into Primary Cilia Using Semi-Permeabilized Cells

    PubMed Central

    Breslow, David K.; Nachury, Maxence V.

    2016-01-01

    The primary cilium is a protrusion from the cell surface that serves as a specialized compartment for signal transduction. Many signaling factors are known to be dynamically concentrated within cilia and to require cilia for their function. Yet protein entry into primary cilia remains poorly understood. To enable a mechanistic analysis of soluble protein entry into cilia, we developed a method for semi-permeabilization of mammalian cells in which the plasma membrane is permeabilized while the ciliary membrane remains intact. Using semi-permeabilized cells as the basis for an in vitro diffusion-to-capture assay, we uncovered a size-dependent diffusion barrier that restricts soluble protein exchange between the cytosol and the cilium. The manipulability of this in vitro system enabled an extensive characterization of the ciliary diffusion barrier and led us to show that the barrier is mechanistically distinct from those at the axon initial segment and the nuclear pore complex. Because semi-permeabilized cells enable a range of experimental perturbations that would not be easily feasible in intact cells, we believe this methodology will provide a unique resource for investigating primary cilium function in development and disease. PMID:25837393

  3. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.

    PubMed

    Macnaughton, S J; O'Donnell, A G; Embley, T M

    1994-10-01

    The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

  4. A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells.

    PubMed

    Kawamata, Hibiki; Starkov, Anatoly A; Manfredi, Giovanni; Chinopoulos, Christos

    2010-12-01

    We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg(2+)] reported by an Mg(2+)-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg(2+). In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF(3)(-) and sodium orthovanadate (Na(3)VO(4)) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F(1)F(o)-ATPase due to its sensitivity to BeF(3)(-) and Na(3)VO(4). With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.

  5. 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

  6. 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.

  7. Actions of antidiuretic hormone analogues on intact and nystatin-permeabilized frog skins.

    PubMed

    Jared, Silviya Rajakumari; Rao, J Prakasa; Subramani, Sathya

    2009-12-01

    The roles of two antidiuretic hormone analogues, namely arginine vasotocin (AVT) and lysine vasopressin (LVP), in solute transport across the ventral abdominal skin of frogs (Rana hexadactyla) were studied using voltage-clamp methods on intact and nystatin-permeabilized preparations. Arginine vasotocin (40 nm), the amphibian analogue of antidiuretic hormone, did not have any effect on the skin of Rana hexadactyla. However, LVP, the porcine antidiuretic hormone, increased the transepithelial potential difference (TEPD) and short-circuit current (SCC) significantly, without affecting the slope conductance. Lysine vasopressin had no action subsequent to addition of amiloride (100 microm) on the apical side or ouabain (10 microm) on the basolateral side. Lysine vasopressin increased slope conductance in the nystatin-permeablized skin while decreasing TEPD. Such a change was not seen in chloride-free solutions. To elucidate the mechanism of action of LVP on intact skin, experiments were done with forskolin and a V(2) receptor blocker. The effects of forskolin (10 microm) were different from those of LVP in that forskolin significantly increased SCC and conductance of the intact skin, while decreasing TEPD. The forskolin-induced increase in conductance was not abolished by amiloride. Use of the V(2) receptor blocker inhibited the effects of LVP. We conclude that AVT does not have an action on the skin of Rana hexadactyla. Lysine vasopressin enhances transepithelial sodium transport by increasing sodium-potassium pump activity, while not affecting the epithelial sodium channel conductance. Lysine vasopressin also enhances an inward-directed conductance on the basolateral membrane, probably a chloride conductance. The action of LVP on the intact frog skin is through the V(2) receptors; however, downstream signalling does not seem to be mediated by cAMP. Analysis of the electrophysiological model of frog skin with LVP allows us additionally to conclude that modulation of

  8. 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.

  9. Effects of fullerenol nanoparticles on acetamiprid induced cytoxicity and genotoxicity in cultured human lung fibroblasts.

    PubMed

    Çavaş, Tolga; Çinkılıç, Nilüfer; Vatan, Özgür; Yılmaz, Dilek

    2014-09-01

    The aim of this study was to investigate the effects of water soluble fullerene (fullerenol) nanoparticles on the in vitro genotoxicity induced by the insecticide acetamiprid. Healthy human lung cells (IMR-90) were treated with fullerenol C60(OH)n (n: 18-22) alone and in combination with acetamiprid for 24h. The micronucleus test, comet assay and γ-H2AX foci formation assays were used as genotoxicity endpoints. Cytotoxicity was evaluated using the clonogenic assay. The maximum tested concentration of fullerenol (1.600 μg/ml) induced 77% survival where as the lowest concentration (25 μg/ml) was not cytotoxic where as acetamiprid was cytotoxic. Fullerenol did not induce genotoxicity at tested concentrations (50-1600 μg/L). On the other hand, acetamiprid (>50 μM) significantly induced formation of micronuclei, and double and single stranded DNA breaks in IMR-90 cells. For simultaneous exposure studies, two non-cytotoxic concentrations (50 and 200 μg/ml) of fullerenol and three cytotoxic concentrations of acetamiprid (100, 200 and 400 μM) were selected. As a result, we observed that co-exposure with fullerenol significantly reduced the cytotoxicity and genotoxicity of acetamiprid in IMR-90 cells. Our results indicated the protective effect of water soluble fullerene particles on herbicide induced genotoxicity.

  10. 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.

  11. The Effect of Botulinum Toxin Type A on Expression Profiling of Long Noncoding RNAs in Human Dermal Fibroblasts

    PubMed Central

    Miao, Ying-ying; Liu, Juan; Zhu, Jie; Tao, Yan-ling; Zhang, Jia-an

    2017-01-01

    Objective. This study was aimed at analyzing the expressions of long noncoding RNAs (lncRNAs) in Botulinum Toxin Type A (BoNTA) treated human dermal fibroblasts (HDFs) in vitro. Methods. We used RNA sequencing to characterize the lncRNAs and mRNAs transcriptome in the control and BoNTA treated group, in conjunction with application of GO (gene ontology) analysis and KEGG (kyoto encyclopedia of genes and genomes) analysis to delineate the alterations in gene expression. We also obtained quantitative real time polymerase chain reaction (qRT-PCR) to confirm some differentially expressed genes. Results. Numerous differentially expressed genes were observed by microarrays between the two groups. qRT-PCR confirmed the changes of six lncRNAs (RP11-517C16.2-001, FR271872, LOC283352, RP11-401E9.3, FGFR3P, and XXbac-BPG16N22.5) and nine mRNAs (NOS2, C13orf15, FOS, FCN2, SPINT1, PLAC8, BIRC5, NOS2, and COL19A1). Farther studies indicated that the downregulating effect of BoNTA on the expression of FGFR3P was time-related and the dosage of BoNTA at a range from 2.5 U/106 cells to 7.5 U/106 cells increased the expression of FGFR3P and COL19A1 in HDFs as well. Conclusion. The expression profiling of lncRNAs was visibly changed in BoNTA treated HDFs. Further studies should focus on several lncRNAs to investigate their functions in BoNTA treated HDFs and the underlying mechanisms. PMID:28265570

  12. 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.

  13. 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

    2014-09-05

    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.

  14. Exploring the effects of low-level laser therapy on fibroblasts and tumor cells following gamma radiation exposure.

    PubMed

    Ramos Silva, Camila; Cabral, Fernanda Viana; de Camargo, Claudinei Francisco Morais; Núñez, Silvia Cristina; Mateus Yoshimura, Tania; de Lima Luna, Arthur Cássio; Maria, Durvanei Augusto; Ribeiro, Martha Simões

    2016-12-01

    Ionizing radiation (IR) induces DNA damage and low-level laser therapy (LLLT) has been investigated to prevent or repair detrimental outcomes resulting from IR exposure. Few in vitro studies, however, explore the biological mechanisms underlying those LLLT benefits. Thus, in this work, fibroblasts and tumor cells are submitted to IR with doses of 2.5 Gy and 10 Gy. After twenty-four-h, the cells are exposed to LLLT with fluences of 30 J cm(-2) , 90 J cm(-2) , and 150 J cm(-2) . Cellular viability, cell cycle phases, cell proliferation index and senescence are evaluated on days 1 and 4 after LLLT irradiation. For fibroblasts, LLLT promotes - in a fluence-dependent manner - increments in cell viability and proliferation, while a reduction in the senescence was observed. Regarding tumor cells, no influences of LLLT on cell viability are noticed. Whereas LLLT enhances cell populations in S and G2 /M cell cycle phases for both cellular lines, a decrease in proliferation and increase in senescence was verified only for tumor cells. Putting together, the results suggest that fibroblasts and tumor cells present different responses to LLLT following exposure to gamma-radiation, and these promising results should stimulate further investigations. Senescence of tumor cells and fibroblasts on the 4(th) day after ionizing radiation (IR) and low-level laser therapy (LLLT) exposures. The number of senescent cells increased significantly for tumor cells (a) while for fibroblasts no increment was observed (b). The blue collor indicates senescence activity.

  15. Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in mouse embryonic fibroblasts.

    PubMed

    Lee, Yu-Hsuan; Cheng, Fong-Yu; Chiu, Hui-Wen; Tsai, Jui-Chen; Fang, Chun-Yong; Chen, Chun-Wan; Wang, Ying-Jan

    2014-05-01

    With the advancement of nanotechnology, nanomaterials have been comprehensively applied in our modern society. However, the hazardous impacts of nanoscale particles on organisms have not yet been thoroughly clarified. Currently, there exist numerous approaches to perform toxicity tests, but common and reasonable bio-indicators for toxicity evaluations are lacking. In this study, we investigated the effects of silver nanoparticles (AgNPs) on NIH 3T3 cells to explore the potential application of these nanoparticles in consumer products. Our results demonstrated that AgNPs were taken up by NIH 3T3 cells and localized within the intracellular endosomal compartments. Exposure to AgNPs is a potential source of oxidative stress, which leads to the induction of reactive oxygen species (ROS), the up-regulation of Heme oxygenase 1 (HO-1) expression, apoptosis and autophagy. Interestingly, AgNPs induced morphological and biochemical markers of autophagy in NIH 3T3 cells and induced autophagosome formation, as evidenced by transmission electron microscopic analysis, the formation of microtubule-associated protein-1 light chain-3 (LC3) puncta and the expression of LC3-II protein. Thus, autophagy activation may be a key player in the cellular response against nano-toxicity.

  16. The effect of vancomycin powder on human dural fibroblast culture and its implications for dural repair during spine surgery.

    PubMed

    Goldschmidt, Ezequiel; Rasmussen, Jorge; Chabot, Joseph D; Gandhoke, Gurpreet; Luzzi, Emilia; Merlotti, Lina; Proni, Romina; Loresi, Mónica; Hamilton, D Kojo; Okonkwo, David O; Kanter, Adam S; Gerszten, Peter C

    2016-11-01

    OBJECTIVE Surgical site infections (SSIs) are a major source of morbidity after spinal surgery. Several recent studies have described the finding that applying vancomycin powder to the surgical bed may reduce the incidence of SSI. However, applying vancomycin in high concentrations has been shown in vitro to inhibit osteoblast proliferation and to induce cell death. Vancomycin may have a deleterious effect on dural healing after repair of an intentional or unintentional durotomy. This study was therefore undertaken to assess the effect of different concentrations of vancomycin on a human dura mater cell culture. METHODS Human dura intended for disposal after decompressive craniectomy was harvested. Explant primary cultures and subcultures were subsequently performed. Cells were characterized through common staining and immunohistochemistry. A growth curve was performed to assess the effect of different concentrations of vancomycin (40, 400, and 4000 μg/ml) on cell count. The effect of vancomycin on cellular shape, intercellular arrangement, and viability was also evaluated. RESULTS All dural tissue samples successfully developed into fusiform cells, demonstrating pseudopod projections and spindle formation. The cells demonstrated vimentin positivity and also had typical features of fibroblasts. When applied to the cultures, the highest dose of vancomycin induced generalized cell death within 24 hours. The mean (± SD) cell counts for control, 40, 400, and 4000 μg/ml were 38.72 ± 15.93, 36.28 ± 22.87, 19.48 ± 6.53, and 4.07 ± 9.66, respectively (p < 0.0001, ANOVA). Compared with controls, vancomycin-exposed cells histologically demonstrated a smaller cytoplasm and decreased pseudopodia formation resulting in the inhibition of normal spindle intercellular arrangement. CONCLUSIONS When vancomycin powder is applied locally, dural cells are exposed to a concentration several times greater than when delivered systemically. In this in vitro model, vancomycin induced

  17. 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

  18. 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

  19. 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

  20. Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts.

    PubMed

    Cavalcanti, Bruno C; Júnior, Hélio V N; Seleghim, Mirna H R; Berlinck, Roberto G S; Cunha, Geanne M A; Moraes, Manoel O; Pessoa, Claudia

    2008-08-11

    Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 microg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 microg/mL). After 24h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50>100 microg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations

  1. Effect of transforming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican.

    PubMed Central

    Romarís, M; Bassols, A; David, G

    1995-01-01

    We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7544118

  2. Turbulent states and their transitions in mathematical models for ventricular tissue: the effects of random interstitial fibroblasts.

    PubMed

    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 G_{j} and (2) the number N_{f} 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) G_{j} is large or (b) the maximum possible number of fibroblasts per myocyte N_{f}^{max} is large. We also observe that the minimum value of G_{j}, for the transition from the ST to the RS state, decreases as N_{f}^{max} 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) G_{j} increases, with fixed N_{f}^{max}, and (2) N_{f}^{max} increases, with fixed G_{j}. We obtain the boundary between ST and RS stability regions in the N_{f}^{max}-G_{j} plane. In particular, for low values of N_{f}^{max}, the value of G_{j}, at the ST-RS boundary, depends on the realization of the randomly distributed fibroblasts; this dependence decreases as N_{f}^{max} 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

  3. Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes.

    PubMed Central

    Bleasdale, J E; Thakur, N R; Rader, G R; Tesan, M

    1985-01-01

    Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+-free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo-inositol that were found previously to be associated with this period of development. PMID:3004409

  4. 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

  5. Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

    PubMed

    Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

    2013-11-01

    In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced

  6. EFFICACY AND POTENCY OF CLASS I ANTIARRHYTHMIC DRUGS FOR SUPPRESSION OF Ca2+ WAVES IN PERMEABILIZED MYOCYTES LACKING CALSEQUESTRIN

    PubMed Central

    Galimberti, Eleonora Savio; Knollmann, Bjorn C.

    2011-01-01

    Background Ca2+ waves can trigger ventricular arrhythmias such as catecholaminergic-polymorphic ventricular tachycardia (CPVT). Drugs that prevent Ca2+ waves may have antiarrhythmic properties. Here, we use permeabilized ventricular myocytes from a CPVT mouse model lacking calsequestrin (casq2) to screen all clinically available class I antiarrhythmic drugs and selected other antiarrhythmic agents for activity against Ca2+ waves. Methods and Results Casq2−/− myocytes were imaged in line-scan mode and the following Ca2+ wave parameters analyzed: wave incidence, amplitude, frequency, and propagation speed. IC50 (potency) and maximum inhibition (efficacy) were calculated for each drug. Drugs fell into 3 distinct categories. Category 1 drugs (flecainide, R-propafenone) suppressed wave parameters with the highest potency (IC50 < 10 μM) and efficacy (> 50% maximum wave inhibition). Category 2 drugs (encainide, quinidine, lidocaine, verapamil) had intermediate potency (IC50 20 μ 40 μM) and efficacy (20% - 40% maximum wave inhibition). Category 3 drugs (procainamide, disopyramide, mexilitine, cibenzoline, ranolazine) had no significant effects on Ca2+ waves at the highest concentration tested (100 μM). Propafenone was stereoselective, with R-propafenone suppressing waves more potently than S-propafenone (IC50: R-propafenone 2±0.2 μM vs. S-propafenone 54±18 μM). Both flecainide and R-propafenone decreased Ca2+ spark mass and converted propagated Ca2+ waves into non-propagated wavelets and frequent sparks, suggesting that reduction in spark mass, not spark frequency, was responsible for wave suppression. Conclusions Among all class I antiarrhythmic drugs, flecainide and R-propafenone inhibit Ca2+ waves with the highest potency and efficacy. Permeabilized casq2−/− myocytes are a simple in-vitro assay for finding drugs with activity against Ca2+ waves. PMID:21798265

  7. 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

  8. 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.

  9. 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.

  10. Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells.

    PubMed

    Sand, Sverre L; Oppegård, Camilla; Ohara, Shinya; Iijima, Toshio; Naderi, Soheil; Blomhoff, Heidi K; Nissen-Meyer, Jon; Sand, Olav

    2010-07-01

    Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca(2+) imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10-100 microM PlnA, whereas concentrations below 10 microM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.

  11. Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

    PubMed

    Jäättelä, Marja; Nylandsted, Jesper

    2015-11-02

    Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

  12. Effect of Cinacalcet and Vitamin D Analogs on Fibroblast Growth Factor-23 during the Treatment of Secondary Hyperparathyroidism

    PubMed Central

    Wetmore, James B.; Gurevich, Konstantin; Da Roza, Gerald; Buerkert, John; Reiner, Maureen; Goodman, William; Cooper, Kerry

    2015-01-01

    Background and objectives Cinacalcet and vitamin D are often combined to treat secondary hyperparathyroidism (SHPT) in patients on dialysis. Independent effects on fibroblast growth factor-23 (FGF-23) concentrations in patients on hemodialysis administered cinacalcet or vitamin D analogs as monotherapies during treatment of SHPT are evaluated. Design, setting, participants, & measurements A multicenter, randomized, open-label study to compare the efficacy of cinacalcet versus traditional vitamin D therapy for management of secondary hyperparathyroidism among subjects undergoing hemodialysis (PARADIGM) was a prospective, phase 4, multicenter, randomized, open-label study conducted globally. Participants (n=312) were randomized 1:1 to cinacalcet (n=155) or vitamin D analog (n=157) for 52 weeks. Levels of FGF-23 were measured at baseline and weeks 20 and 52. The absolute and percentage changes from baseline in plasma FGF-23, parathyroid hormone (PTH), calcium (Ca), phosphorus (P), and calcium-phosphorus product (Ca×P) were assessed. Correlations and logistic regression were used to explore relationships between changes in FGF-23 and changes in PTH, Ca, P, and Ca×P from baseline to week 52 by treatment arm. Results Median (quartiles 1, 3) decrease in FGF-23 concentrations was observed in the cinacalcet arm (−40%; −63%, 16%) compared with median increase in the vitamin D analog arm (47%; 0%, 132%) at week 52 (P<0.001). Changes in FGF-23 in both arms were unrelated to changes in PTH (cinacalcet: r=0.17, P=0.11; vitamin D analog: r=−0.04, P=0.70). Changes in FGF-23 in the vitamin D analog but not the cinacalcet arm were correlated with changes in Ca (cinacalcet: r=0.11, P=0.30; vitamin D analog: r=0.32, P<0.01) and P (cinacalcet: r=0.19, P=0.07; vitamin D analog: r=0.49, P<0.001). Changes in FGF-23 were correlated with changes in Ca×P in both arms (cinacalcet: r=0.26, P=0.01; vitamin D analog: r=0.57, P<0.001). Independent of treatment arm, participants with

  13. 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.

  14. Structure activity relationship of antioxidative property of flavonoids and inhibitory effect on matrix metalloproteinase activity in UVA-irradiated human dermal fibroblast.

    PubMed

    Sim, Gwan-Sub; Lee, Bum-Chun; Cho, Ho Seung; Lee, Jae Woong; Kim, Jin-Hwa; Lee, Dong-Hwan; Kim, Jin-Hui; Pyo, Hyeong-Bae; Moon, Dong Cheul; Oh, Ki-Wan; Yun, Yeo Pyo; Hong, Jin Tae

    2007-03-01

    Collagenase, a matrix metalloproteinases (MMPs), is a key regulator in the photoaging process of skin due to the reactive oxygen species generated after exposure to ultraviolet A (UVA). Flavonoid compounds have been demonstrated to possess antioxidant properties, and could be useful in the prevention of photoaging. In this study, to investigate the structure-activity relationship of flavonoid compounds on their antioxidant property and inhibitory effects against the MMP activity, the effects of several flavonoids; myricetin, quercetin, kaempferol, luteolin, apigenin and chrysin, on the reactive oxygen species scavengering activity and inhibitory effect against the MMP activity were examined in vitro and in human dermal fibroblasts induced by UVA. The relative order of antioxidative efficacy, as determined using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method and the xanthine/xanthine oxidase system, was as follows; flavones: luteolin > apigenin > chrysin, flavonols: myricetin > quercetin > kaempferol, and correlated with the respective number of OH group on their B-ring. In good correlation with the antioxidant properties, the flavonoids inhibited the collagenase activities, in a dose-dependent manner, and the MMP expression. These results suggested the UVA induced antioxidative activity and inhibitory effects of flavonoids on the collagenase in human dermal fibroblasts depends on the number of OH group in the flavonoid structure, and those with a higher number of OH group may be more useful in the prevention of UV stressed skin aging.

  15. Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

    NASA Astrophysics Data System (ADS)

    Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

    2010-02-01

    Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

  16. 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.

  17. 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

  18. 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

    Ho, Manh Tin; Kang, Hyun Sik; Huh, Jung Sik; Kim, Young Mee; Lim, Yoongho; Cho, Moonjae

    2014-07-23

    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.

  19. 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

  20. Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins.

    PubMed

    Arias, Mauricio; Quijano, Jairo C; Haridas, Valsala; Gutterman, Jordan U; Lemeshko, Victor V

    2010-06-01

    Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the "osmotic protection" method with 100mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.

  1. 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.

  2. A lack of synergy between membrane-permeabilizing cationic antimicrobial peptides and conventional antibiotics.

    PubMed

    He, Jing; Starr, Charles G; Wimley, William C

    2015-01-01

    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.

  3. Structural Correlates of Antibacterial and Membrane-Permeabilizing Activities in Acylpolyamines

    PubMed Central

    Balakrishna, Rajalakshmi; Wood, Stewart J.; Nguyen, Thuan B.; Miller, Kelly A.; Suresh Kumar, E. V. K.; Datta, Apurba; David, Sunil A.

    2006-01-01

    A homologous series of mono- and bis-acyl polyamines with varying acyl chain lengths originally synthesized for the purpose of sequestering lipopolysaccharide were evaluated for antimicrobial activity to test the hypothesis that these bis-cationic amphipathic compounds may also bind to and permeabilize intact gram-negative bacterial membranes. Some compounds were found to possess significant antimicrobial activity, mediated via permeabilization of bacterial membranes. Structure-activity relationship studies revealed a strong dependence of the acyl chain length on antimicrobial potency and permeabilization activity. Homologated spermine, bis-acylated with C8 or C9 chains, was found to profoundly sensitize Escherichia coli to hydrophobic antibiotics such as rifampin. Nonspecific cytotoxicity is a potential drawback of these membranophilic compounds. However, the surface activity of these cationic amphipaths is strongly attenuated under physiological conditions via binding to serum albumin. Significant antibacterial activity is still retained in the presence of physiological concentrations of human serum albumin, suggesting that these compounds may serve as leads in the development of novel adjuncts to conventional antimicrobial chemotherapy. PMID:16495242

  4. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    PubMed

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  5. Inhibitory effects of C-type natriuretic peptide on the differentiation of cardiac fibroblasts, and secretion of monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1

    PubMed Central

    LI, ZHI-QIANG; LIU, YING-LONG; LI, GANG; LI, BIN; LIU, YANG; LI, XIAO-FENG; LIU, AI-JUN

    2015-01-01

    The present study aimed to investigate the effect of C-type natriuretic peptide (CNP) on the function of cardiac fibroblasts (CFs). Western blotting was used to investigate the expression of myofibroblast marker proteins: α-smooth muscle actin (α-SMA), extra domain-A fibronectin, collagen I and collagen III, and the activity of extracellular signal-regulated kinase 1/2 (ERK1/2). Immunofluorescence was used to examine the morphological changes; a transwell assay was used to analyze migration, and reverse transcription-quantitative polymerase chain reaction and ELISA were employed to determine the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). The results demonstrated that CNP significantly reduced the protein expression of α-SMA, fibronectin, collagen I and collagen III, and suppressed the migratory ability of CFs. Additionally, the mRNA and protein expression of MCP-1 and PAI-1 was inhibited under the CNP treatment; and this effect was mediated by the inhibition of the ERK1/2 activity. In conclusion, CNP inhibited cardiac fibroblast differentiation and migration, and reduced the secretion of MCP-1 and PAI-1, which demonstrates novel mechanisms to explain the antifibrotic effect of CNP. PMID:25352084

  6. Promising anti-growth effects of palladium(II) saccharinate complex of terpyridine by inducing apoptosis on transformed fibroblasts in vitro.

    PubMed

    Coskun, Melis Debreli; Ari, Ferda; Oral, Arzu Yilmaztepe; Sarimahmut, Mehmet; Kutlu, Hatice M; Yilmaz, Veysel T; Ulukaya, Engin

    2013-08-01

    Fibrosarcoma is one of the fatal cancer types and there is still not satisfactory success in its treatment despite new drugs. Therefore, the search for a new compound has been going on. It is currently known that some palladium-based anti-cancer compounds seem to have powerful apoptosis-inducing effects in cancer cells. For this purpose, a palladium(II)-saccharinate complex containing terpyridine which was synthesized by our research group was investigated in terms of its anti-tumor effects against mouse embryonic fibroblast NIH/3T3 (normal cell line) and rat embryonic fibroblast 5RP7 (H-ras transformed cell line) in vitro. The MTT and ATP viability assays were used to determine anti-growth/cytotoxic effects. Cytotoxic activity was confirmed by real time cytotoxicity analysis system. Flow cytometry analysis was further used to determine the mode of cell death (apoptosis/necrosis). Apoptosis was confirmed by triple-staining the cells with Hoechst 33342/PI/Calcein-AM triple and evaluated with fluorescence microscopy. It was found that the compound showed significant anti-growth activity by inducing apoptosis in a dose dependent manner. In conclusion, taking into account the cytotoxic activity of the compound at even relatively lower doses, in vivo experiments to elucidate its potential use for the treatment of fibrosarcoma are warranted.

  7. Walking dead: Permeabilization of heat-treated Geobacillus stearothermophilus ATCC 12980 spores under growth-preventing conditions.

    PubMed

    Mtimet, Narjes; Trunet, Clément; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

    2017-06-01

    Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth.

  8. 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

  9. 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.

  10. Effect of postactivation treatment with latrunculin A on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged Clawn miniature boar.

    PubMed

    Himaki, Takehiro; Mizobe, Yamato; Tsuda, Kenichirou; Suetomo, Masashi; Yamakuchi, Hiroshi; Miyoshi, Kazuchika; Takao, Sonshin; Yoshida, Mitsutoshi

    2012-01-01

    The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.

  11. Effects of licarin E on expression of matrix metalloproteinase-1 and type-1 procollagen in UVB-irradiated human skin fibroblasts.

    PubMed

    Kwon, Yi-Young; Kim, Daeyoung; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-12-01

    Ultraviolet B (UVB) radiation induces photoaging by upregulating the expression of matrix metalloproteinase (MMP) and decreasing collagen synthesis in human skin cells. This study evaluated the effects of licarin E isolated from mace, the aril of Myristica fragrans Houtt., on MMP-1 and type-1 procollagen levels in UVB-irradiated human skin fibroblasts. Powdered mace extracted with 95% ethanol was used and licarin E isolated by preparative high-performance liquid chromatography. In addition, western blot analysis, reverse transcription PCR and electrophoretic mobility shift assay were used to evaluate the effects of licarin E and its molecular mechanism. It was found that licarin E attenuated UVB-induced MMP-1 expression by inactivating mitogen-activated protein kinases (MAPKs), thereby inhibiting activator protein 1. Licarin E also increased type-1 procollagen expression by stimulating transforming growth factor β (TGFβ)/Smad signaling. The findings show that licarin E positively regulates the expression of MMP-1 and type-1 procollagen in UVB-irradiated human skin fibroblasts through MAPK and TGFβ signaling, suggesting its potential as a potent antiphotoaging agent.

  12. 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.

  13. Magainin 2a - Induced Permeabilization of Phospholipid Vesicles

    DTIC Science & Technology

    1991-01-02

    unilamellar vesicles (SUVs) of phosphatidylserine ( PS ). Addition of peptide to the SUVs causes an initial rapid release of dye, lasting about 100 sec...Measurements 39 VII. Results 40 A. Effect of MGN2a on the Permeability of PS Vesicles 40 B. Effect of Extravesicular Osmotic Pressure on 6CF Release from...Depiction of all-or-none versus graded release mechanisms 34 4. Standard quenching curve for 6CF entrapped within PS SUVs 38 5. MGN2a-induced release

  14. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.

    PubMed

    Bewley, Martin A; Naughton, Michael; Preston, Julie; Mitchell, Andrea; Holmes, Ashleigh; Marriott, Helen M; Read, Robert C; Mitchell, Timothy J; Whyte, Moira K B; Dockrell, David H

    2014-10-07

    Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY's ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. Importance: Streptococcus

  15. Plasma Membrane Permeabilization by Trains of Ultrashort Electric Pulses

    DTIC Science & Technology

    2009-01-01

    ultrashort electrical pulses. Bioelectromagnetics , 2001. 22(6): p. 440-8. 4. Stacey, M., et al., Differential effects in cells exposed to ultra-short...nanosecond pulsed electric field Bioelectromagnetics , 2007. 28: p. 655-663. 13. Gowrishankar, T.R. and J.C. Weaver, Electrical behavior and pore...electric field (nsPEF). Bioelectromagnetics , 2007. 28: p. 655- 663. 19. Nuccitelli, R., et al., A new pulsed electric field therapy for melanoma disrupts

  16. The Effect of a Novel c.820C>T (Arg274Trp) Mutation in the Mitofusin 2 Gene on Fibroblast Metabolism and Clinical Manifestation in a Patient

    PubMed Central

    Kawalec, Maria; Kabzińska, Dagmara; Kochański, Andrzej; Krzyśko, Krystiana A.; Zabłocka, Barbara

    2017-01-01

    Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a de novo Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions. PMID:28076385

  17. Effects of macelignan isolated from Myristica fragrans (Nutmeg) on expression of matrix metalloproteinase-1 and type I procollagen in UVB-irradiated human skin fibroblasts.

    PubMed

    Lee, Kyung-Eun; Mun, Sukyeong; Pyun, Hee-Bong; Kim, Myung-Suk; Hwang, Jae-Kwan

    2012-01-01

    Exposure to ultraviolet (UV) light causes premature skin aging that is associated with upregulated matrix metalloproteinases (MMPs) and decreased collagen synthesis. Macelignan, a natural lignan compound isolated from Myristica fragrans HOUTT. (nutmeg), has been reported to possess antioxidant and antiinflammatory activities. This study assessed the effects of macelignan on photoaging and investigated its mechanisms of action in UV-irradiated human skin fibroblasts (Hs68) by reverse transcription-polymerase chain reaction, Western blot analysis, 2',7'-dichlorofluorescein diacetate assay, and enzyme-linked immunosorbent assay. Our results show that macelignan attenuated UV-induced MMP-1 expression by suppressing phosphorylation of mitogen-activated protein kinases (MAPKs) induced by reactive oxygen species. Macelignan also increased type I procollagen expression and secretion through transforming growth factor β (TGF-β)/Smad signaling. These findings indicate that macelignan regulates the expression of MMP-1 and type I procollagen in UV-irradiated human skin fibroblasts by modulating MAPK and TGF-β/Smad signaling, suggesting its potential as an efficacious antiphotoaging agent.

  18. 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.

  19. Contribution of Fibroblast and Mast Cell (Afferent) and Tumor (Efferent) IL-6 Effects within the Tumor Microenvironment.

    PubMed

    Hugo, Honor J; Lebret, Stephanie; Tomaskovic-Crook, Eva; Ahmed, Nuzhat; Blick, Tony; Newgreen, Donald F; Thompson, Erik W; Ackland, M Leigh

    2012-04-01

    Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a 'wound that never heals'. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal

  20. 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

  1. Chromosome fragility in New Zealand black mice: effect of ultraviolet and gamma radiations on fetal fibroblasts in vitro

    SciTech Connect

    Reddy, A.L.; Fialkow, P.J.

    1980-04-01

    The sensitivity of fibroblasts cultured from New Zealand Black (NZB) and BALB/c mouse fetuses to uv and gamma radiations was tested with two methods: (1) colony-forming ability and (2) chromosome abnormalities. When compared with BALB/c cells, NZB cells had reduced colony-forming ability and increased chromosome abnormalities after uv irradiation. However, no differences were seen in colony formation or frequency of chromosome abnormalities between NZB and BALB/c cells after exposure to gamma radiation. This apparent uv specificity strengthens the suggestion that NZB mice might be used as a model to study the relationship between chromosome abnormalities and cancer in human syndromes such as xeroderma pigmentosum, which is characterized by chromosome instability.

  2. Mechanism of membrane permeabilization by sticholysin I, a cytolysin isolated from the venom of the sea anemone Stichodactyla helianthus.

    PubMed

    Tejuca, M; Serra, M D; Ferreras, M; Lanio, M E; Menestrina, G

    1996-11-26

    Actinaria cytolysins are very potent basic toxins isolated from the venom of sea anemones, which are supposed to exert their toxic activity through formation of oligomeric pores in the host plasma membrane. To gain insight into their mechanism of action, the interaction of Stichodactyla helianthus sticholysin I (St-I) with lipid bilayers was studied. St-I increased the permeability of calcein-loaded lipid vesicles composed of different phospholipids. The rate of permeabilization improved when sphingomyelin (SM) was introduced into phosphatidylcholine (PC) vesicles, reaching an optimum value at equimolar concentrations of these two phospholipids. It was also a function of the pH, showing a local maximum of activity between pH 8 and 9 and a marked decrease at pH 10 and 11. Under optimal conditions (e.g., PC:SM 1:1, pH 8, toxin to vesicle ratio < 200), most of the toxin is bound to the lipid phase. The reduced toxin effect at low and high SM content, or at high pH, is principally due to a decreased toxin binding. From the dose dependence of the permeabilization, at constant lipid concentration, it was inferred that St-I increases membrane permeability by forming oligomeric pores comprising at least three cytolysin monomers. The involvement of oligomers was also suggested by the dependence of calcein release on the vesicle concentration at constant toxin dose. In fact, the time course of dye release was well described under all circumstances by a kinetic model which assumes that trimerization leads to a conductive pore. All the relevant equilibrium and rate constants were derived. Addition of St-I to one side of a planar lipid membrane increased the conductivity of the film in discrete steps of defined amplitude, indicating the formation of ion channels. The dose dependence of this effect was the same as with LUV. The channel was cation-selective and its conductance suggested a functional radius of about 1.0 nm, consistent with the size of the lesion previously

  3. 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.

  4. 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

    Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

    2010-06-01

    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 H(2)O(2)-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 H(2)O(2)-induced cell damage in vitro.

  5. Gaining myocytes or losing fibroblasts: Challenges in cardiac fibroblast reprogramming for infarct repair.

    PubMed

    Nagalingam, Raghu S; Safi, Hamza A; Czubryt, Michael P

    2016-04-01

    Unlike most somatic tissues, the heart possesses a very limited inherent ability to repair itself following damage. Attempts to therapeutically salvage the myocardium after infarction, either by sparing surviving myocytes or by injection of exogenous cells of varied provenance, have met with limited success. Cardiac fibroblasts are numerous, resistant to hypoxia, and amenable to phenotype reprogramming to cardiomyocytes - a potential panacea to an intractable problem. However, the long-term effects of mass conversion of fibroblasts are as-yet unknown. Since fibroblasts play key roles in normal cardiac function, treating these cells as a ready source of replacements for myocytes may have the effect of swapping one problem for another. This review briefly examines the roles of cardiac fibroblasts, recaps the strides made so far in their reprogramming to cardiomyocytes both in vitro and in vivo, and discusses the potential ramifications of large-scale cellular identity swapping. While such therapy offers great promise, the potential repercussions require consideration and careful study.

  6. DNA excision repair in permeable human fibroblasts

    SciTech Connect

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the (3H)DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells.

  7. 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

  8. 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

  9. Membrane permeabilization induced by Triton X-100: The role of membrane phase state and edge tension.

    PubMed

    Mattei, Bruno; Lira, Rafael B; Perez, Katia R; Riske, Karin A

    2017-01-01

    Detergents are widely used to solubilize and separate biomembrane components. It is therefore relevant to study and understand the mechanistic details underlying detergent-lipid interactions using biomimetic systems. Here, we have investigated in detail the process of membrane permeabilization and the nature of pores induced by sub-solubilizing concentrations of the detergent Triton X-100 (TX-100) in bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), sphingomyelin (SM) and binary mixtures of these phospholipids with 30 mol% cholesterol (chol). A fluorescence quenching assay was used to evaluate the permeability of large unilamellar vesicles (LUVs) in the presence of increasing concentrations of TX-100. Confocal microscopy was employed to visualize and quantify the permeability of giant unilamellar vesicles (GUVs) to two fluorescent dyes of different sizes in the presence of TX-100. Both methods showed that POPC, POPC/chol and SM membranes become fully permeable at a specific TX-100 concentration, followed by complete (POPC and SM) and partial (POPC/chol) solubilization at a higher detergent concentration. The confocal microscopy experiments revealed that opening of pores occurs as a well-defined event and that for POPC and POPC/chol the pores were initially selective to the small probe and then grew and allowed passage of the larger dye as well. On the other hand, the insoluble SM/chol membranes exhibited only a mild TX-100-induced permeabilization. The membrane edge tension of the liquid phases was measured from the closure rate of macropores induced by electric pulses in GUVs. Membrane edge tension was shown to be sensitive to membrane composition and to decrease in the presence of TX-100. We propose that extensive permeabilization occurs below a critical membrane edge tension, which is eventually reached in the partially and fully soluble compositions, but not in the insoluble mixture.

  10. 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.

  11. Anti-syntaxin antibodies inhibit calcium-dependent catecholamine secretion from permeabilized chromaffin cells.

    PubMed

    Gutierrez, L M; Quintanar, J L; Viniegra, S; Salinas, E; Moya, F; Reig, J A

    1995-01-05

    Adrenomedullary chromaffin cells release catecholamines in response to the intracellular calcium rise upon stimulation by different secretagogues. The presence of syntaxin 1, a protein presumably involved in docking of synaptic vesicles to presynaptic membranes, has been investigated in chromaffin cells. The study using two different monoclonal antibodies shows that syntaxin 1 is present in the chromaffin cell membrane fraction. Functional experiments demonstrate that anti-syntaxin antibodies inhibit calcium-dependent secretion in permeabilized cells. These results suggest that syntaxin 1 is an important component of the secretory machinery in chromaffin cells.

  12. Hsp90 regulation of fibroblast activation in pulmonary fibrosis

    PubMed Central

    Sontake, Vishwaraj; Wang, Yunguan; Kasam, Rajesh K.; Sinner, Debora; Reddy, Geereddy B.; Naren, Anjaparavanda P.; McCormack, Francis X.; Jegga, Anil G.; Madala, Satish K.

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β–driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease. PMID:28239659

  13. PINOCYTOSIS IN FIBROBLASTS

    PubMed Central

    Steinman, Ralph M.; Silver, Jonathan M.; Cohn, Zanvil A.

    1974-01-01

    Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 106 cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q10 was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown. PMID:4140194

  14. Novel real-time cell analysis platform for the dynamic monitoring of ionizing radiation effects on human tumor cell lines and primary fibroblasts.

    PubMed

    Mán, Imola; Szebeni, Gábor J; Plangár, Imola; Szabó, Emilia R; Tőkés, Tünde; Szabó, Zoltán; Nagy, Zoltán; Fekete, Gábor; Fajka-Boja, Roberta; Puskás, László G; Hideghéty, Katalin; Hackler, László

    2015-09-01

    Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a real‑time label‑free assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedance‑based platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT‑29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional end‑point cell‑based methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescence‑activated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The real‑time data correlated with the colony forming assay results. At low cell densities (1,000‑2,000 cells/well) the impedance‑based method was more accurate at monitoring dose‑d