Cultured Mouse Fibroblasts Research Articles

The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules

The inflammatory response plays out over time in a reproducible and organized manner after an initiating stimulus. Here we showed that the genes activated in cultured mouse fibroblasts in response to the cytokine tumor necrosis factor (TNF) can be divided roughly into three groups, each with different induction kinetics. Whereas differential transcription is important in determining the grouping of these genes, differential mRNA stability also exerted a strong influence—in some cases overriding that of transcriptional control elements—on the temporal order of gene expression. mRNA transcripts expressed early have abundant AU-rich elements in their 3′-untranslated regions whereas those expressed later contain fewer. Thus mRNA stability and transcriptional control, two intrinsic characteristics of genes, control the kinetics of proinflammatory cytokine-induced gene expression.

シンポジウム３‐１ 中枢神経系の再生・次なる半世紀 ｉＰＳ細胞技術と神経疾患研究

Induced pluripotent stem (iPS) cells are pluripotent stem cells directly reprogrammed from cultured mouse fibroblast by introducing Oct3/4, Sox2, c-Myc, and Klf4. Cells obtained using this technology, which allows the ethical issues and immunological rejection associated with embryonic stem (ES) cells to be avoided, might be a clinically useful source for cell replacement therapies. We found that secondary neurospheres (SNSs) generated from various mouse iPS cell showed their neural differentiation capacity and teratoma formation after transplantation into the brain of immunodeficient NOD/SCID mice. We found that origin (source of somatic cells) of the iPS cells are the crucial determinant for the potential tumorigenicity of iPS-derived neural stem/progenitor cells and that their tumorigenicity results from the persistent presence of undifferentiated cells within the SNSs. Surprisingly, SNSs derived from c-Myc minus iPS cells generated without drug selection showed robust tumorigenesis, in spite of their potential to contribute adult chimeric mice without tumor formation. Furthermore, we examined whether the transplantation of non-tumorigenic Nanog-iPS-derived SNSs into mouse spinal cord injury (SCI) model could promote locomotor function recovery. As a result, we found that properly pre-evaluated iPS clone-derived neural stem/progenitor cells may be a promising cell source for future transplantation therapy of SCI.

Preparation of Gel Film fromBombyx moriSilk Sericin and Its Characterization as a Wound Dressing

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable beta-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.

In vitro evaluation of cytotoxicity of soft lining materials on L929 cells by MTT assay

The aim of this in vitro study was to evaluate five commonly used soft lining materials, Viscogel (VG), Ufi Gel P (UGP), Softliner (S), Coe-Soft (CS), and Molloplast-B (MB) in terms of cytotoxicity by MTT assay, using L929 mouse fibroblasts. Sixteen disk-shaped specimens from each material were prepared (according to the manufacturer's instructions) in stainless steel mold (10 mm diameter and 1.5 mm thick). The specimens were incubated for 24, 48, 72, and 96 h in Dulbecco's Modified Eagle Medium Ham's F12 (DMEM/F12) and following each incubation interval, cytotoxicity of the extracts to cultured mouse fibroblasts (L929) were measured by MTT assay. Data were statistically analyzed by two-way analysis of variance (ANOVA), and Duncan's test, at a significance level of p < 0.05. Group CS revealed significantly high cytotoxic effect at all incubation periods (p < 0.05). Although no cytotoxic effect for Group S was found at 24, 48, 72 h periods, it has been raised at 96-h incubation period (p > 0.05). Group VG, UGP, S (except at 96 h period), and Group MB demonstrated high cell survival rates at incubation periods.

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773–15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.

Fibrillin-1 microfibril deposition is dependent on fibronectin assembly

Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of beta1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of beta1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectin(RGE/RGE) mutant mouse fibroblast cultures, which do not engage alpha5beta1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

Low cytotoxicity of creams and lotions formulated with Buriti oil ( Mauritia flexuosa) assessed by the neutral red release test

The aim of this work was to evaluate possible cytotoxic effects of topical creams and lotions produced with Buriti oil and commercial surfactants on human keratinocytes HaCat and 3T3 embryonic mouse fibroblast cultures. We also aimed to assess the cytotoxicity of the surfactants used to produce the emulsions. The neutral red release (NRR) assay was performed as an in vitro method to evaluate the cytotoxicity of the emulsions in HaCat and 3T3 cell lines and predict potential skin irritation. The Buriti oil emulsions presented low cytotoxicity to the cells at high concentrations and the addition of Vitamin E increased cell viability. Among the surfactant tested, Unitol ® CE 200F proved to be the most cytotoxic, presenting an IC 50 significantly lower than the others. Emulsions formulated with Buriti oil and commercial surfactants could be non irritant to the skin due to their low cytotoxicity, especially when enhanced with vitamin E. When emulsified with Buriti oil, water and Brij 72, Unitol CE200F showed less cytotoxic effects than when tested alone.

Activation of Cyclic AMP Signaling in Ae2-deficient Mouse Fibroblasts

Anion exchanger 2 (AE2, SLC4A2) is a ubiquitously expressed membrane solute carrier that regulates intracellular pH (pH(i)) by exchanging cytosolic bicarbonate for extracellular chloride. We used fibroblasts from Ae2-deficient (Ae2(a,b)(-/-)) mice to study the effects of an alkaline shift in resting intracellular pH (pH(i)) on the activation of cAMP signaling and gene expression. Ae2(a,b)(-/-) fibroblasts show increased pH(i) (by 0.22 +/- 0.03 unit) compared with wild type cells at extracellular pH (pH(o)) 7.4 and 37 degrees C. This shift in resting pH(i) is associated with an up-regulation of bicarbonate-activated soluble adenylyl cyclase expression, increased cAMP production, Creb phosphorylation, inducible cAMP early repressor 1 mRNA expression, and impaired activation of c-Fos transcription by forskolin. These results highlight the importance of bicarbonate transport via Ae2 in maintaining pH(i) homeostasis in cultured mouse fibroblasts and unveil the role of cAMP in the cellular response to chronic alkalization, which putatively includes an inducible cAMP early repressor 1-mediated attenuation of phosphorylated Creb activity.

The role of kinases in the Hedgehog signalling pathway

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

Micro-and nanostructural characteristics of 3D porous carriers ElastoPHB®-3D

Microstructure of the surface and micro- and nanostructure of the internal surface of 3D porous carrier ElastoPHB-3D were studied by methods of electron microscopy and atomic force microscopy. Biological properties of ElastoPHB-3D samples were evaluated using culture of L929 mouse fibroblasts. High porosity and pore size of biodegradable matrixes ElastoPHB-3D and their good biofunctional properties as the substrate for cell culturing allow us to recommend ElastoPH-3D as a promising carrier for cell transplantation and creation of artificial organs.

Telomeres Acquire Distinct Heterochromatin Characteristics during siRNA-Induced RNA Interference in Mouse Cells

Telomeres are protective structures present at the ends of linear chromosomes and consist of simple repeating-DNA sequences and specialized proteins [1, 2]. Integrity of the telomeres is important in maintaining genome stability[1-6]. RNA interference(RNAi) involves short double-stranded RNA (21-23 nucleotides long), termed short interference RNA(siRNA), resulting in the downregulation of genes with cognate sequences [7-9]. During transient siRNA-induced RNAi in mouse fibroblast cultures, we found significant reversible changes related to the telomeres. Telomeres acquired distinct heterochromatin features. There were increased bindings of Argonaute-1 (AGO1), telomeric repeat-binding factor 1(TERF1), and heterochromatin protein 1beta (HP1beta) on the telomeres. Histone H3 (lysine 9) was hypermethylated at the telomeres. The chromosome ends also were associated with an unidentified RNA. During RNAi, expression of a transgene inserted adjacent to the telomere was downregulated. In addition, the concentration of a group of heterogeneous high-molecular-weight RNA containing telomeric repeat sequences was increased, and this RNA formed a small number of transient, discrete nuclear foci. Our findings suggest that telomeres participate actively in the siRNA-induced RNAi process. These responses of telomeres to the RNAi process might partially account for the off-target effects of RNAi.

Uptake of a Recombinant Human .ALPHA.-L-Iduronidase (laronidase) by Cultured Fibroblasts and Osteoblasts

To examine the uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts from a patient with mucopolysaccharidosis I (MPS I) and its effect on the cleavage of accumulated substrates, we performed enzymological, Western blotting, immunocytochemical and morphological studies. Laronidase was incorporated into the MPS I cells dose-dependently mainly via mannose 6-phosphate (M6P) receptors. Then the incorporated enzyme was transported to lysosomes and processed to the mature form, the pathological changes of the cells being improved. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts. The enzyme was incorporated into the cultured mouse osteoblasts mainly via M6P receptors, although mannose (Man) receptors were partially involved in the uptake of the enzyme, as in the cultured fibroblasts. But the uptake by the former was apparently lower than that by the latter. The administration of a high dose of the enzyme or development of a recombinant alpha-L-iduronidase containing many M6P residues is required for further improvement of enzyme replacement therapy for skeletal disorders caused by MPS I.

Induction of pluripotent stem cells from fibroblast cultures

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Abstract 150: Novel Mechanism for Regulation of Extracellular SOD Expression and Activity by Antioxidant-1

Extracellular superoxide dismutase (ecSOD), a copper-containing secretory antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Little is known regarding the mechanisms by which ecSOD expression and activity are regulated. We previously demonstrated that antioxidant-1 (Atox1) functions as a copper chaperone for ecSOD at transGolgi network (TGN) to regulate its activity. Unexpectedly, we also found that ecSOD mRNA is markedly decreased in Atox1-knockout (KO) fibroblasts. We thus hypothesize that Atox1 functions not only as copper chaperone but also as transcription factor to regulate both expression and activity of ecSOD. Here we show that in ApoE-KO mouse aorta, Atox1 was intensely stained in the nucleus at the intimal lesion of atherosclerosis where ecSOD was highly expressed. In cultured mouse fibroblast, copper treatment stimulated translocation of Atox1 from the cytosol to the nucleus. The ecSOD mRNA and its promoter activity were markedly decreased in Atox1 KO cells (82.5% and 85.7% decrease, respectively). Promoter deletion analysis identified Atox1-response element (Atox1-RE) at −314/−304 segment of ecSOD promoter. Gel shift and ChIP assays showed that Atox1 directly bound to the Atox1-RE including DNA segment in a copper dependent manner. To segregate a transcription factor and a copper chaperone function of Atox1, we generated adenovirus expressing wild-type, nucleus-targeted, and TGN-targeted Atox1 (Atox1-WT, Atox1-NLS, and Atox1-TGN). Re-expression of either Atox1-NLS or Atox1-WT, but not Atox1-TGN, in Atox1-KO cells increased ecSOD mRNA by 3.2-and 2.5-fold, respectively. Importantly, re-expression of either Atox1-WT only or both Atox1-NLS and Atox1-TGN in Atox1 KO cells increased ecSOD activity (2.1- and 2.7-fold, respectively), while either Atox1-NLS or Atox1-TGN alone had no effect. In summary, Atox1 functions as a novel copper dependent transcription factor to increase ecSOD expression, and modulates ecSOD activity through both copper chaperone and transcription factor function. Thus, not only ecSOD but also Atox1 are potential therapeutic targets for oxidant stress-dependent cardiovascular disease such as atherosclerosis.

Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

Metabolic alterations in cultured mouse fibroblasts induced by an inhibitor of the tyrosine kinase receptor Fibroblast Growth Factor Receptor 1

Proton nuclear magnetic resonance (NMR) spectroscopy was used to identify and quantify the metabolites present in cultured mouse fibroblast cells 3T6 in their native state and after treatment with PD166866, an inhibitor of the fibroblast growth factor receptor. Cell extracts were prepared according to the Bligh–Dyer protocol which prevents artifacts deriving from the chemical demolition of macromolecules. Also the growth medium was subjected to the same extraction procedure. The NMR approach made possible the identification and quantification of about 40 different metabolites at nanomoles/mg of protein level: the biological relevance of the variation of some metabolite levels is discussed. Our experimental procedure offers a prospective method for the evaluation of variations of the metabolic profile deriving from different biochemical treatments of these cells.

Bioactive Metabolites from the Caribbean Sponge Aka coralliphagum

The chemistry of the burrowing sponge Aka coralliphagum was investigated to identify chemically labile secondary metabolites. The HPLC-MS analysis of the two growth forms typica and incrustans revealed different metabolites. The previously unknown sulfated compounds siphonodictyals B1 to B3 (6-8), corallidictyals C (9) and D (10), and siphonodictyal G (11) were isolated, and their structures were elucidated by NMR and MS experiments. The compounds were tested in a DPPH assay, in antimicrobial assays against bacteria, yeasts, and fungi, and in antiproliferation assays using cultures of mouse fibroblasts. The biological activity was linked to the presence of the ortho-hydroquinone moiety.

Semiautomated Multiplexed Quantum Dot-Based in Situ Hybridization and Spectral Deconvolution

Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.

Effect of light curing type on cytotoxicity of dentine‐bonding agents

To compare the cytotoxic effects of dentine-bonding agents (DBAs) polymerized with two different curing units at 24 h and 72 h on L-929 cells. Disc-shaped test samples of light-activated DBAs were prepared according to manufacturers' instructions and cured with either conventional quartz tungsten halogen or light-emitting diode light curing units (LCUs). After curing, the samples were transferred into a culture medium for 24 h. Eluates were obtained and pipetted onto L-929 mouse fibroblast cultures (3 x 10(4) cells per well), incubated for evaluation after 24 and 72 h. After both incubation periods, measurements were performed by an dimethylthiazol diphenyltetrazolium assay. The degree of cytotoxicity for each sample was determined according to the reference value represented by the cells with a control (culture without sample). Statistical significance was determined by a three-way analysis of variance followed by the Mann-Whitney U-test. No significant three-factor interaction occurred amongst LCUs, DBAs and time factors (P = 0.955). LCUs and DBAs had a significant two-factor interaction (P < 0.001). In general, the test materials cured with the light-emitting diode LCU demonstrated higher cell survival rates when compared with the those cured with the quartz tungsten halogen. Differential toxic effects of the DBAs cured with the quartz tungsten halogen or the light-emitting diode on the fibroblast cells may prove to be very important when suitable DBAs or LCUs are used for operative restorations.

Drug Binding and Releasing Characteristics of DNA/Lipid/PLGA Film

We evaluated the blended compound of DNA/lipid complexes and PLGA (poly(D,L-lactide-co-glycolide)) as a carrier material for drug delivery system (DDS). Transparent, self-standing DNA/lipid/PLGA films were prepared by casting from an organic solvent such as DMSO/chloroform. Daunorubicin hydrochloride (DH) could intercalate and groove bind into DNA in the films, whereby the amount of DH bound to the films was controlled by the latter's immersion period in DH aqueous solution. DH was released from DH films after immersion in PBS solution, whereby release rate was dependent on the chemical structure of lipids. Released DH caused reduction of cell viability during the cell culture of L929 mouse fibroblasts. These results suggested that DNA/lipid/PLGA film was a promising useful material for DDS.