Gelsolin, a Protein That Caps the Barbed Ends and Severs Actin Filaments, Enhances the Actin-Based Motility ofListeria monocytogenes in Host Cells

ABSTRACT The actin-based motility of Listeria monocytogenesrequires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected withListeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean ± standard error of the mean, 0.09 ± 0.003 μm/s [n = 176] versus 0.05 ± 0.003 μm/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases inListeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellularListeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

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Effect of heat treatment on cytotoxicity of self-adhesive resin cements: Cell viability analysis

European Journal of Dentistry ◽

10.4103/ejd.ejd_34_18 ◽

2018 ◽

Vol 12 (02) ◽

pp. 281-286 ◽

Cited By ~ 3

Author(s):

Celso Afonso Klein-Júnior ◽

Roberto Zimmer ◽

Guilherme Scotta Hentschke ◽

Denise Cantarelli Machado ◽

Rubem Beraldo dos Santos ◽

...

Keyword(s):

Heat Treatment ◽

Cell Viability ◽

Fibroblast Cell ◽

Resin Cements ◽

Adhesive Resin ◽

Viability Analysis ◽

3T3 Fibroblasts ◽

Mixing Control ◽

Nih 3T3

ABSTRACT Objective: The aim of the study was to assess, in vitro, the influence on cytotoxicity of heat treatment applied before photopolymerization, while mixing three self-adhesive resin cements, in an NIH/3T3 fibroblast cell culture, based on cell viability measures. Methods: Samples were divided into three groups: (1) no heat treatment while mixing (control), (2) 37°C, and (3) 60°C heat treatment while mixing. Cements were light-cured immediately after mixing and immersed in Dulbecco's Modified Eagle Media for the extraction of possibly uncured products after 24 h and 7 days. Cultures contained 0.5 mL of NIH/3T3 fibroblasts per well at a concentration of 0.4 × 105 cells/mL and specific extracts for each sample. Statistical Analysis Used: Data were statistically analyzed with ANOVA and post hoc Student–Newman–Keuls (significance of 5%). Results: Cement cytotoxicity increased with time, as shown by the higher values observed at 7 days. There was a slight difference in intragroup cytotoxicity levels between 24 h and 7 days. Heat treatment at 60°C was associated with a major decrease in cytotoxicity levels in all three groups, both at 24 h and at 7 days, with no differences among the cements. Conclusions: Heat treatment at 60°C should be considered as a strategy to reduce cytotoxicity of self-adhesive resin cements, as evidenced by the results observed at 24 h and 7 days of analysis.

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Alteration of Fibroblast Cell Behavior due to Contraction of Substrate

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19507 ◽

2010 ◽

Author(s):

Uday Chippada ◽

Xue Jiang ◽

Michelle Previtera ◽

Rene Schloss ◽

Bernard Yurke ◽

...

Keyword(s):

Cell Attachment ◽

Fibroblast Cell ◽

Cellular Responses ◽

Cell Behavior ◽

Cellular Behavior ◽

Polyacrylamide Hydrogels ◽

3T3 Fibroblasts ◽

Nih 3T3

Many researchers have utilized hydrogels as substrates for cell attachment. The stiffness of these substrates has been found to influence the cellular behavior such as morphology, proliferation, growth and differentiation. Lo et al. deformed polyacrylamide substrates with a blunted microneedle and observed the movement of NIH 3T3 fibroblasts. In both pulling and pushing, the cells reversed their direction and moved away from the needle. This shows that cellular behavior is also affected by stretching the underlying substrates. In a previous study, Lin et al. have demonstrated the ability to contract DNA-crosslinked polyacrylamide hydrogels (‘DNA gels’ in short) by addition of crosslinks. Jiang et al. have utilized these DNA gels as substrates to observe the cellular responses of L929 and GFP fibroblasts to both static and dynamic substrate compliances.

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A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

The Journal of Cell Biology ◽

10.1083/jcb.122.2.461 ◽

1993 ◽

Vol 122 (2) ◽

pp. 461-471 ◽

Cited By ~ 47

Author(s):

EK Han ◽

TM Guadagno ◽

SL Dalton ◽

RK Assoian

Keyword(s):

Cell Cycle ◽

Growth Factor ◽

Cell Cycle Progression ◽

Fibroblast Cell ◽

Tgf Beta ◽

Cycle Progression ◽

Anchorage Independent Growth ◽

Differential Effects ◽

3T3 Fibroblasts ◽

Nih 3T3

We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using G0-synchronized NRK and NIH-3T3 cells, we showed that anchorage-independent growth is regulated by an attachment-dependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae. In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion. Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition. In related studies, we show that TGF-beta 1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta 1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta 1 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-beta 1 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-beta 1 on anchorage-independent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S. Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-beta 1 requirement for G1/S transit while retaining their normal mitogen requirements for proliferation. These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited. The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-beta 1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage-independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence.

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A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.1983-1993.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 1983-1993 ◽

Cited By ~ 38

Author(s):

Amardeep S. Dhillon ◽

Sharon Meikle ◽

Carole Peyssonnaux ◽

Joan Grindlay ◽

Christian Kaiser ◽

...

Keyword(s):

Cell Transformation ◽

Primary Cultures ◽

Erk Pathway ◽

Morphological Alterations ◽

Extracellular Signal Regulated Kinase ◽

Amino Terminal ◽

3T3 Fibroblasts ◽

Extracellular Signal ◽

Accelerated Proliferation ◽

Nih 3T3

ABSTRACT It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

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Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

International Journal of Molecular Sciences ◽

10.3390/ijms23010509 ◽

2022 ◽

Vol 23 (1) ◽

pp. 509

Author(s):

Jinho Park ◽

Pavlo Kravchuk ◽

Adithi Krishnaprasad ◽

Tania Roy ◽

Ellen Hyeran Kang

Keyword(s):

Actin Filament ◽

Chemical Properties ◽

Fibroblast Cell ◽

Cellular Level ◽

Dependent Manner ◽

Cellular Functions ◽

Concentration Dependent Manner ◽

Filament Dynamics ◽

Assembly Kinetics ◽

Nih 3T3

Actin plays critical roles in various cellular functions, including cell morphogenesis, differentiation, and movement. The assembly of actin monomers into double-helical filaments is regulated in surrounding microenvironments. Graphene is an attractive nanomaterial that has been used in various biomaterial applications, such as drug delivery cargo and scaffold for cells, due to its unique physical and chemical properties. Although several studies have shown the potential effects of graphene on actin at the cellular level, the direct influence of graphene on actin filament dynamics has not been studied. Here, we investigate the effects of graphene on actin assembly kinetics using spectroscopy and total internal reflection fluorescence microscopy. We demonstrate that graphene enhances the rates of actin filament growth in a concentration-dependent manner. Furthermore, cell morphology and spreading are modulated in mouse embryo fibroblast NIH-3T3 cultured on a graphene surface without significantly affecting cell viability. Taken together, these results suggest that graphene may have a direct impact on actin cytoskeleton remodeling.

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Wound healing potentials of a herbal homeopathic remedy on NIH 3T3 fibroblasts in vitro

Planta Medica ◽

10.1055/s-0031-1282787 ◽

2011 ◽

Vol 77 (12) ◽

Author(s):

K Hostanska ◽

M Rostock ◽

R Saller

Keyword(s):

Wound Healing ◽

Homeopathic Remedy ◽

3T3 Fibroblasts ◽

Nih 3T3

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Self-Targeting of Carbon Dots into the Cell Nucleus: Diverse Mechanisms of Toxicity in NIH/3T3 and L929 Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22115608 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5608

Author(s):

Markéta Havrdová ◽

Iztok Urbančič ◽

Kateřina Bartoň Tománková ◽

Lukáš Malina ◽

Janez Štrancar ◽

...

Keyword(s):

Dna Damage ◽

Carbon Dots ◽

L929 Cell ◽

Fluorescent Properties ◽

Cell Cycle Profile ◽

Mechanisms Of Toxicity ◽

3T3 Fibroblasts ◽

Nih 3T3 ◽

Positively Charged ◽

Endocytic Pathways

It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.

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Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis

Human Reproduction ◽

10.1093/humrep/dez109 ◽

2019 ◽

Vol 34 (8) ◽

pp. 1536-1550 ◽

Cited By ~ 2

Author(s):

A Christine Kauerhof ◽

Nour Nicolas ◽

Sudhanshu Bhushan ◽

Eva Wahle ◽

Kate A Loveland ◽

...

Keyword(s):

Protein Expression ◽

Activin A ◽

Collagen I ◽

3T3 Cells ◽

Qrt Pcr ◽

3T3 Fibroblasts ◽

Total Collagen ◽

Severity Of The Disease ◽

Nih 3T3 ◽

Nih 3T3 Cells

Abstract STUDY QUESTION Does activin A contribute to testicular fibrosis under inflammatory conditions? SUMMARY ANSWER Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. WHAT IS KNOWN ALREADY Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. STUDY DESIGN, SIZE, DURATION This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined. Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7). Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P < 0.01, P < 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P < 0.05), accompanied by increased total collagen (P < 0.01) and fibronectin deposition. Total testicular collagen (P < 0.0001) and fibronectin protein expression (P < 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r = 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P < 0.05) and the production of collagen I (P < 0.001; P < 0.01) and fibronectin (P < 0.05). Moreover, activin A also increased collagen IV mRNA (P < 0.05) in PTC, while αSMA mRNA (P < 0.01) and protein (P < 0.0001) were significantly increased by activin A in NIH 3T3 cells. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. WIDER IMPLICATIONS OF THE FINDINGS Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government’s Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1–2) on `Molecular pathogenesis on male reproductive disorders’ funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

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In VitroAntileukemic Activity ofXanthosoma sagittifolium(Taioba) Leaf Extract

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/384267 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Marina L. C. Caxito ◽

Rachell R. Correia ◽

Anne Caroline C. Gomes ◽

Graça Justo ◽

Marsen G. P. Coelho ◽

...

Keyword(s):

Antitumor Activity ◽

Leaf Extract ◽

Leukemia Cells ◽

No Production ◽

3T3 Fibroblasts ◽

Griess Reaction ◽

Xanthosoma Sagittifolium ◽

M Phase ◽

Nih 3T3

Xanthosoma sagittifoliumSchott is a herb of the Araceae family, popularly known as taioba, which is consumed as food in some regions of Brazil, Africa, and Asia. This species has already been evaluated for the antifungal activities. However, based on its potential antitumor activity, the present study further aimed to examine the antitumor, as well as chelation, activity ofX. sagittifoliumleaf extract. Results showed that hydroethanolic extract ofX. sagittifoliumleaves (HEXs-L) exhibits cytotoxic effects against the immortalized line of human T-lymphocytic (Jurkat) and myelogenous (K562) leukemia cells, but not nontumor RAW 264.7 macrophages or NIH/3T3 fibroblasts. HEXs-L inhibited 50.3% of Jurkat cell proliferation, reducing by 20% cells in G2/M phase, but increasing cells in sub-G1 phase, thereby inducing apoptosis by 54%. In addition, HEXs-L inhibited NO production by 59%, as determined by Griess reaction, and chelated 93.8% of free Fe(II), as demonstrated by ferrozine assay. Phytochemical studies were carried out by ESI-MS, identifying apigenin di-C-glycosides as major compounds. Overall, this work revealed that leaf extract ofXanthosoma sagittifoliumpresented chelating activity andin vitroantitumor activity, arresting cell cycle and inducing apoptosis of leukemia cells, thus providing evidence that taioba leaves may have practical application in cancer therapy.

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